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Intra-Electron Transfer Induced by Protonation in Copper-Containing Nitrite Reductase
Abstract
The inter- and intra-electron, and proton transfers in the nitrite reduction of copper-containing nitrite reductase (CuNiR) were investigated by using QM/MM method with the calculational models containing type 1 (T1) and type2 (T2) Cu sites. The electron transfer from the outer electron donor protein to T1 Cu site occurred at the timing of both before and after the nitrite binding, and the nitrite binding lowered the reduction potential of Cu T1 site. The protonation of catalytic His244 subsequent to the nitrite binding and T1 Cu reduction induced partial intra-electron transfer from T1 to T2 Cu sites. The proton transfer from His244 to nitrite bound on T2 Cu site via the hydrogen bond network induced intra-electron transfer from T1 to T2 Cu site. The interaction of T1 Cu ligand with the second sphere amino acid residues and water molecules affected the reduction potential of T1 Cu site. The water molecules in so-called proton pool have an important role in regulation the basicity of His244. The conformation of sensor loop did not change along the reaction, but the water molecule network extending along the sensor loop was changed by the nitrite biding.
... [26,59] Crystal structure of CuNiRs shows that His260 is localized in the interface of two monomers and contacts with His255 through two H 2 Os (W H1 , W H2 ) in a socalled proton pool, which was proposed to play an important role in modulating the protonation state of His255. [27,28,63] Therefore, to reduce the computational cost, we examined the process of PT from His260 to His255 utilizing the new model of H260À W H2 À H255. The PT from His260 to His255 is mediated by two inserted H 2 Os, including two elementary steps. ...
... These results are in agreement with the view of the Lintuluoto group that proton migrating around the T2Cu site can cause intra-ET from T1Cu to T2Cu sites. [59,63] However, Lintuluoto, et al. proposed that the protonation of His244 (His255 in 1AS6 of AfNiR) induces partial electron migrate from T1Cu to T2Cu and PT from Asp98 to the nitrite substrate causes more electron transfer from T1Cu to T2Cu. [63] Our examinations revealed that the driving force of intra-ET comes from the remote watermediated triple-proton transfer along the both proton channels. ...
... [59,63] However, Lintuluoto, et al. proposed that the protonation of His244 (His255 in 1AS6 of AfNiR) induces partial electron migrate from T1Cu to T2Cu and PT from Asp98 to the nitrite substrate causes more electron transfer from T1Cu to T2Cu. [63] Our examinations revealed that the driving force of intra-ET comes from the remote watermediated triple-proton transfer along the both proton channels. These results are consistent with the recent proposal of PT along the long water hydrogen bond network coupled ET in CuNiRs by experimental methods. ...
The copper‐containing nitrite reductase (CuNiR) catalyzes the biological conversion of nitrite to nitric oxide; key long‐range electron/proton transfers are involved in the catalysis. However, the details of the electron‐/proton‐transfer mechanism are still unknown. In particular, the driving force of the electron transfer from the type‐1 copper (T1Cu) site to the type‐2 copper (T2Cu) site is ambiguous. Here, we explored the two possible proton‐transfer channels, the high‐pH proton channel and the primary proton channel, by using two‐layered ONIOM calculations. Our calculation results reveal that the driving force for electron transfer from T1Cu to T2Cu comes from a remote water‐mediated triple‐proton‐coupled electron‐transfer mechanism. In the high‐pH proton channel, the water‐mediated triple‐proton transfer occurs from Glu113 to an intermediate water molecule, whereas in the primary channel, the transfer is from Lys128 to His260. Subsequently, the two channels employ another two or three distinct proton‐transfer steps to deliver the proton to the nitrite substrate at the T2Cu site. These findings explain the detailed proton‐/electron‐transfer mechanisms of copper‐containing nitrite reductase and could extend our understanding of the diverse proton‐coupled electron‐transfer mechanisms in complicated proteins.
... 26 The protonation states of these residues on NO 2 − binding are elusive and require further investigation. However, two protons are required for the turnover of NO 2 − to NO so we use the common hypothesis that the proton is transferred from Asp CAT to an NO 2 − oxygen, 23 and the Asp CAT could in turn be protonated by His CAT or from water in the water channel. Rather than providing the explicit pathway of proton transfer necessary for the reduction, we are using His CAT as a possible proton donor to Asp CAT. 23 The crystal water that bridges Asp CAT and His CAT via a hydrogen bond participates in the proton relay from His CAT to Asp CAT . ...
... However, two protons are required for the turnover of NO 2 − to NO so we use the common hypothesis that the proton is transferred from Asp CAT to an NO 2 − oxygen, 23 and the Asp CAT could in turn be protonated by His CAT or from water in the water channel. Rather than providing the explicit pathway of proton transfer necessary for the reduction, we are using His CAT as a possible proton donor to Asp CAT. 23 The crystal water that bridges Asp CAT and His CAT via a hydrogen bond participates in the proton relay from His CAT to Asp CAT . Guided by the MSOX data, we started with the side-on NO 2 − structure, bound to Cu(I) according to our modeling, and optimized it after protonation of the nitrite, without altering the initial protonation states of Asp CAT and His CAT (the system used is Asp98p-Hsp) ( Figure 6). ...
The recently developed multiple structures from one crystal (MSOX) serial crystallography method can be used to provide multiple snapshots of the progress of enzymatic reactions taking place within a protein crystal. Such MSOX snapshots can be used as a reference for combined quantum mechanical/molecular mechanical (QM/MM) simulations of enzyme reactivity within the crystal. QM/MM calculations are used to identify details of reference states that cannot be directly observed by X-ray diffraction experiments, such as protonation and oxidation states. These reference states are then used as known fixed endpoints for the modeling of reaction paths. We investigate the mechanism of nitrite reduction in an Achromobacter cycloclastes copper nitrite reductase crystal using MSOX-guided QM/MM calculations, identifying the change in nitrite binding orientation with a change in copper oxidation state, and determining the reaction path to the final NO-bound MSOX structure. The results are compared with QM/MM simulations performed in a solvated environment.
... [5][6][7][8][9][10][11] The reduction mechanism of copper nitrite reductase (CuNiR) is still under debate due to conflicting evidence between crystallographic, spectroscopic and computational data. 8,9,11,[16][17][18][19][20][21] Studies agree that nitrite binding, intramolecular electron transfer (ET) from T1-Cu to T2-Cu, proton transfer (PT) to nitrite, and product release are all involved in nitrite reduction. However, questions surrounding the protonation states of secondary shell residues Asp98 and His255 and the mechanism on how intramolecular ET occurs remain unresolved. ...
... 12 The former case was suggested in recent experimental studies, 13,14 while most of previous computational studies treat intramolecular ET and PT as separate processes. [16][17][18][19] Existing studies have already established the importance of Asp98 and His255 in the reduction mechanism of CuNiR based on the pH of optimal activity, which is between 5 to 7, which is closely correlated to the pH dependence of intramolecular ET in nitrite-bound CuNiR. 24,25 Mutagenesis studies also show that mutations of these two residues result in substantially decreased enzymatic activity. ...
Nitrite reductases are enzymes that aid in the denitrification process by catalyzing the reduction of nitrite to nitric oxide gas. Since this reaction is the first committed step that involves gas formation, it is regarded to be a vital step for denitrification. However, the mechanism of copper-containing nitrite reductase is still under debate due to discrepancy between theoretical and experimental data, especially in terms of the roles of secondary shell residues Asp98 and His255 and the electron transfer mechanism between two copper sites. Herein, we revisited the nitrite reduction mechanism of A. faecalis copper nitrite reductase using QM(B3LYP)/MM-based metadynamics. It is found that the intramolecular electron transfer from T1-Cu to T2-Cu occurs via an asynchronous proton-coupled electron transfer (PCET) mechanism, with electron transfer (ET) preceding proton transfer (PT). In particular, we found that the ET process is driven by the conformation conversion of Asp98 from gatekeeper to proximal one, which is much more energy-demanding than the PCET itself. These results highlight that the inclusion of electron donor is vital in investigating the electron-transfer related processes like PCET
... The catalytic mechanism of NirK involves Asp and His residues that donate two protons necessary for nitrite to nitric oxide conversion. These residues are conserved in Haloferax mediterranei NirK, being His 278 and Asp 132 (Figure 6A), and the proposed mechanism [70,[76][77][78] could also take place in NirK from Haloferax mediterranei. The physiological electron donors to NirK in Haloferax mediterranei is still unknown. ...
During the last century, anthropogenic activities such as fertilization have led to an increase in pollution in many ecosystems by nitrogen compounds. Consequently, researchers aim to reduce nitrogen pollutants following different strategies. Some haloarchaea, owing to their denitrifier metabolism, have been proposed as good model organisms for the removal of not only nitrate, nitrite, and ammonium, but also (per)chlorates and bromate in brines and saline wastewater. Bacterial denitrification has been extensively described at the physiological, biochemical, and genetic levels. However, their haloarchaea counterparts remain poorly described. In previous work the model structure of nitric oxide reductase was analysed. In this study, a bioinformatic analysis of the sequences and the structural models of the nitrate, nitrite and nitrous oxide reductases has been described for the first time in the haloarchaeon model Haloferax mediterranei. The main residues involved in the catalytic mechanism and in the coordination of the metal centres have been explored to shed light on their structural characterization and classification. These results set the basis for understanding the molecular mechanism for haloarchaeal denitrification, necessary for the use and optimization of these microorganisms in bioremediation of saline environments among other potential applications including bioremediation of industrial waters.
... However, it has been suggested that His CAT has a more relevant role than Asp CAT in the pH-dependent activity, and that the hydrogen bond network of water molecules in the main proton channel, but not the sensing loop, changes its conformation upon nitrite binding. 33 Distinct types of factors have been indicated as possible causes for the lack of activity of NirK at high pH. The X-ray structure of Rhodobacter sphaeroides 2.4.3 ...
Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu²⁺ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu²⁺ LMCT transitions.
... We therefore suggest that the generation of solvated electrons in crystallo by X-ray radiolysis produces a change of the pH in the active-site micro-environment of CuNiRs, shifting the geometry of Asp CAT and therefore affecting the NO 2 À -binding mode. It has been suggested that His CAT has a role as a redox-coupled switch for proton transfer , which is consistent with computational and biophysical studies showing that protonation is required for the rate-limiting intramolecular electron-transfer reaction (Ghosh et al., 2009;Leferink et al., 2011;Lintuluoto & Lintuluoto, 2018). Here, we observed no protonation of N "2 of His CAT at pD 5.4, while the linking water is neutral, suggesting that an internal change in pH is required to transfer the proton from the water (W2) to His CAT . ...
Copper-containing nitrite reductases (CuNiRs) that convert NO 2⁻ to NO via a Cu CAT –His–Cys–Cu ET proton-coupled redox system are of central importance in nitrogen-based energy metabolism. These metalloenzymes, like all redox enzymes, are very susceptible to radiation damage from the intense synchrotron-radiation X-rays that are used to obtain structures at high resolution. Understanding the chemistry that underpins the enzyme mechanisms in these systems requires resolutions of better than 2 Å. Here, for the first time, the damage-free structure of the resting state of one of the most studied CuNiRs was obtained by combining X-ray free-electron laser (XFEL) and neutron crystallography. This represents the first direct comparison of neutron and XFEL structural data for any protein. In addition, damage-free structures of the reduced and nitrite-bound forms have been obtained to high resolution from cryogenically maintained crystals by XFEL crystallography. It is demonstrated that Asp CAT and His CAT are deprotonated in the resting state of CuNiRs at pH values close to the optimum for activity. A bridging neutral water (D 2 O) is positioned with one deuteron directed towards Asp CAT O δ1 and one towards His CAT N ∊2 . The catalytic T2Cu-ligated water (W1) can clearly be modelled as a neutral D 2 O molecule as opposed to D 3 O ⁺ or OD ⁻ , which have previously been suggested as possible alternatives. The bridging water restricts the movement of the unprotonated Asp CAT and is too distant to form a hydrogen bond to the O atom of the bound nitrite that interacts with Asp CAT . Upon the binding of NO 2⁻ a proton is transferred from the bridging water to the O δ2 atom of Asp CAT , prompting electron transfer from T1Cu to T2Cu and reducing the catalytic redox centre. This triggers the transfer of a proton from Asp CAT to the bound nitrite, enabling the reaction to proceed.
Acid-catalyzed conversion of nitrite to nitric oxide at the copper( ii ) center: a new catalytic pathway.
Mononuclear copper complexes relevant to the active site of copper nitrite reductases (CuNiRs) are known to be catalytically active for the reduction of nitrite. Yet, their catalytic mechanism has thus far not been resolved. Here, we provide a complete description of the electrocatalytic nitrite reduction mechanism of a bio-inspired CuNiR catalyst Cu(tmpa) (tmpa = tris(2-pyridylmethyl)amine) in aqueous solution. Through a combination of electrochemical studies, reaction kinetics, and density functional theory (DFT) computations, we show that the protonation steps take place in a stepwise manner and are decoupled from electron transfer. The rate-determining step is a general acid-catalyzed protonation of a copper-ligated nitrous acid (HNO2) species. In view of the growing urge to convert nitrogen-containing compounds, this work provides principal reaction parameters for efficient electrochemical nitrite reduction. This contributes to the investigation and development of nitrite reduction catalysts, which is crucial to restore the biogeochemical nitrogen cycle.
Nitrite reductase (NiR) catalyzes nitrite (NO2 -) to nitric oxide (NO) transformation in the presence of an acid (H+ ions/pH) and serves as a critical step in NO biosynthesis. In addition to the NiR enzyme, NO synthases (NOSs) participate in NO production. The chemistry involved in the catalytic reduction of NO2 -, in the presence of H+, generates NO with a H2O molecule utilizing two H+ + one electron from cytochromes and is believed to be affected by the pH. Here, to understand the effect of H+ ions on NO2 - reduction, we report the acid-induced NO2 - reduction chemistry of a nonheme FeII-nitrito complex, [(12TMC)FeII(NO2 -)]+ (FeII-NO2 -, 2), with variable amounts of H+. FeII-NO2 - upon reaction with one-equiv. of acid (H+) generates [(12TMC)Fe(NO)]2+, {FeNO}7 (3) with H2O2 rather than H2O. However, the amount of H2O2 decreases with increasing equivalents of H+ and entirely disappears when H+ reaches ≅ two-equiv. and shows H2O formation. Furthermore, we have spectroscopically characterized and followed the formation of H2O2 (H+ = one-equiv.) and H2O (H+ ≅ two-equiv.) and explained why bio-driven NiR reactions end with NO and H2O. Mechanistic investigations, using 15N-labeled-15NO2 - and 2H-labeled-CF3SO3D (D+ source), revealed that the N atom in the {Fe14/15NO}7 is derived from the NO2 - ligand and the H atom in H2O or H2O2 is derived from the H+ source, respectively.
In recent years there have been paradigm shifts in our understanding of the diversity of copper-containing nitrite reductases (CuNiRs) found in nature, as genomic mining revealed the widespread distribution of variants with additional tethered redox domains. The availability of these variants is an unparalled opportunity to explore inter-protein electron transfer events that are so important in biology. Access to X-ray free-electron laser sources with femtosecond X-ray pulses has enabled crystal structures of several CuNiRs to be determined free of radiation-induced damage and radiation-induced redox chemistry. By controlling the radiation dose, structural movies of enzyme-bound intermediates of the catalytic cycle have been identified, notably the relevance of a Cu-NO species to catalysis has been established. Such studies have been complemented by an approach using chemically induced turnover of enzyme in crystals. These have allowed damage-free high-resolution, even atomic resolution, structures to be analysed to assign probable protonation states of critical residues complemented by ambient temperature and our neutron crystallographic structure. The determination of X-ray crystal structures at elevated temperatures including room temperature revealed a stable Cu-nitrosyl intermediate during in-crystal turnover. This should enable refinement in computational chemistry modelling and enable a more rational comparison with spectroscopic data relating to solution studies. Using advanced biophysical techniques the link between proton uptake and inter-Cu electron transfer been established. Spectroscopic single-molecule studies have revealed differences in redox behaviour of isolated enzyme compared with turnover. These are significant advances that together with recent refinement of computational models of the CuNiR reaction mechanism are likely to assist in the development of synthetic CuNiRs.
Nitric oxide (NO) is an important signaling molecule that is involved in a wide range of physiological and pathological events in biology. Metal coordination chemistry, especially with iron, is at the heart of many biological transformations involving NO. A series of heme proteins, nitric oxide synthases (NOS), soluble guanylate cyclase (sGC), and nitrophorins, are responsible for the biosynthesis, sensing, and transport of NO. Alternatively, NO can be generated from nitrite by heme- and copper-containing nitrite reductases (NIRs). The NO-bearing small molecules such as nitrosothiols and dinitrosyl iron complexes (DNICs) can serve as an alternative vehicle for NO storage and transport. Once NO is formed, the rich reaction chemistry of NO leads to a wide variety of biological activities including reduction of NO by heme or non-heme iron-containing NO reductases and protein post-translational modifications by DNICs. Much of our understanding of the reactivity of metal sites in biology with NO and the mechanisms of these transformations has come from the elucidation of the geometric and electronic structures and chemical reactivity of synthetic model systems, in synergy with biochemical and biophysical studies on the relevant proteins themselves. This review focuses on recent advancements from studies on proteins and model complexes that not only have improved our understanding of the biological roles of NO but also have provided foundations for biomedical research and for bio-inspired catalyst design in energy science.
Nitric oxide (NO) is the classic non-innocent ligand, and its coordination chemistry has both fascinated and perplexed scientists for the last > 150 years, starting with the discovery of sodium nitroprusside, Na2[Fe(CN)5(NO)], by Playfair in 1849. This is not only due to the fact that this ligand can change oxidation state upon binding to transition metal centers, but also the often highly covalent bonds that originate from this interaction. The coordination chemistry of NO with iron, but also copper and cobalt, then experienced a revival in the 1980s when the biological significance of NO for mammalian physiology was recognized. This article summarizes key accomplishments in the field of the coordination chemistry of NO in the last 10–15 years, and points toward biological relevance wherever appropriate.
Aerobic denitrification is a novel biological nitrogen removal technology, which has been widely investigated as an alternative to the conventional denitrification and for its unique advantages. To fully comprehend aerobic denitrification, it is essential to clarify the regulatory mechanisms of intracellular electron transfer during aerobic denitrification. However, reports on intracellular electron transfer during aerobic denitrification are rather limited. Thus, the purpose of this review is to discuss the molecular mechanism of aerobic denitrification from the perspective of electron transfer, by summarizing the advancements in current research on electron transfer based on conventional denitrification. Firstly, the implication of aerobic denitrification is briefly discussed, and the status of current research on aerobic denitrification is summarized. Then, the occurring foundation and significance of aerobic denitrification are discussed based on a brief review of the key components involved in the electron transfer of denitrifying enzymes. Moreover, a strategy for enhancing the efficiency of aerobic denitrification is proposed on the basis of the regulatory mechanisms of denitrification enzymes. Finally, scientific outlooks are given for further investigation on aerobic denitrification in the future. This review could help clarify the mechanism of aerobic denitrification from the perspective of electron transfer.
It is generally assumed that tethering enhances rates of electron harvesting and delivery to active sites in multi-domain enzymes by proximity and sampling mechanisms. Here, we explore this idea in a tethered 3-domain, trimeric copper-containing nitrite reductase. By reverse engineering, we find that tethering does not enhance the rate of electron delivery from its pendant cytochrome c to the catalytic copper-containing core. Using a linker that harbors a gatekeeper tyrosine in a nitrite access channel, the tethered haem domain enables catalysis by other mechanisms. Tethering communicates the redox state of the haem to the distant T2Cu center that helps initiate substrate binding for catalysis. It also tunes copper reduction potentials, suppresses reductive enzyme inactivation, enhances enzyme affinity for substrate and promotes inter-copper electron transfer. Tethering has multiple unanticipated beneficial roles, the combination of which fine-tunes function beyond simplistic mechanisms expected from proximity and restrictive sampling models.
Copper nitrite reductases (CuNiR) carry out the first committed step of the denitrification pathway of the global nitrogen cycle, the reduction of nitrite (NO2-) to nitric oxide (NO). As such, they are of major agronomic and environmental importance. CuNiRs occur primarily in denitrifying soil bacteria which carry out the overall reduction of nitrate to dinitrogen. In this article, we review the insights gained into copper nitrite reductase function from three dimensional structures. We particularly focus on developments over the last decade, including insights from serial femtosecond crystallography using X-ray free electron lasers (XFELs) and from the recently discovered 3-domain CuNiRs.
We explored the reaction mechanism of the cationic rhodium(I)–BINAP complex catalysed isomerisation of allylic amines using the artificial force induced reaction method with the global reaction route mapping strategy, which enabled us to search for various reaction paths without assumption of transition states. The entire reaction network was reproduced in the form of a graph, and reasonable paths were selected from the complicated network using Prim’s algorithm. As a result, a new dissociative reaction mechanism was proposed. Our comprehensive reaction path search provided rationales for the E/Z and S/R selectivities of the stereoselective reaction.
Amino acid structures are an ideal test set for method-development studies in crystallography. High-resolution X-ray diffraction data for eight previously studied genetically encoding amino acids are provided, complemented by a non-standard amino acid. Structures were re-investigated to study a widely applicable treatment that permits accurate X-H bond lengths to hydrogen atoms to be obtained: this treatment combines refinement of positional hydrogen-atom parameters with aspherical scattering factors with constrained "TLS+INV" estimated hydrogen anisotropic displacement parameters (H-ADPs). Tabulated invariom scattering factors allow rapid modeling without further computations, and unconstrained Hirshfeld atom refinement provides a computationally demanding alternative when database entries are missing. Both should incorporate estimated H-ADPs, as free refinement frequently leads to over-parameterization and non-positive definite H-ADPs irrespective of the aspherical scattering model used. Using estimated H-ADPs, both methods yield accurate and precise X-H distances in best quantitative agreement with neutron diffraction data (available for five of the test-set molecules). This work thus solves the last remaining problem to obtain such results more frequently. Density functional theoretical QM/MM computations are able to play the role of an alternative benchmark to neutron diffraction.
Serial femtosecond crystallography (SFX) has enabled the damage-free structural determination of metalloenzymes and filled
the gaps of our knowledge between crystallographic and spectroscopic data. Crystallographers, however, scarcely know whether
the rising technique provides truly new structural insights into mechanisms of metalloenzymes partly because of limited resolutions.
Copper nitrite reductase (CuNiR), which converts nitrite to nitric oxide in denitrification, has been extensively studied
by synchrotron radiation crystallography (SRX). Although catalytic Cu (T2Cu) of CuNiR had been suspected to tolerate X-ray
photoreduction, we here showed that T2Cu in the form free of nitrite is reduced and changes its coordination structure in
SRX. Moreover, we determined the completely oxidized CuNiR structure at 1.43 Å resolution with SFX. Comparison between the
high-resolution SFX and SRX data revealed the subtle structural change of a catalytic His residue by X-ray photoreduction.
This finding, which SRX has failed to uncover, provides new insight into the reaction mechanism of CuNiR.
Enzyme mechanisms are often probed by structure-informed point mutations and measurement of their effects on enzymatic properties to test mechanistic hypotheses. In many cases, the challenge is to report on complex, often inter-linked elements of catalysis. Evidence for long-range effects on enzyme mechanism resulting from mutations remains sparse, limiting the design/redesign of synthetic catalysts in a predictable way. Here we show that improving the accessibility of the active site pocket of copper nitrite reductase by mutation of a surface-exposed phenylalanine residue (Phe306), located 12 Å away from the catalytic site type-2 Cu (T2Cu), profoundly affects intra-molecular electron transfer, substrate-binding and catalytic activity. Structures and kinetic studies provide an explanation for the lower affinity for the substrate and the alteration of the rate-limiting step in the reaction. Our results demonstrate that distant residues remote from the active site can have marked effects on enzyme catalysis, by driving mechanistic change through relatively minor structural perturbations.
Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO2(-)) to nitric oxide (NO) during denitrification. We determined the crystal structures of CuNIR from thermophilic gram-positive bacterium, Geobacillus thermodenitrificans (GtNIR) in chloride- and formate(-)bound forms of wild type at 1.15 Å resolution and the nitrite-bound form of the C135A mutant at 1.90 Å resolution. The structure of C135A with nitrite displays a unique η(1)-O coordination mode of nitrite at the catalytic copper site (T2Cu), which has never been observed at the T2Cu site in known wild-type CuNIRs, because the mobility of two residues essential to catalytic activity, Asp98 and His244, are sterically restricted in GtNIR by Phe109 on a characteristic loop structure that is found above Asp98 and by an unusually short CH-O hydrogen bond observed between His244 and water, respectively. A detailed comparison of the WT structure with the nitrite-bound C135A structure implies the replacement of hydrogen(-)bond networks around His244 and predicts the flow path of protons consumed by nitrite reduction. Based on these observations, the reaction mechanism of GtNIR through the η(1)-O coordination manner is proposed.
Copper-containing nitrite reductases (CuNiRs), which catalyse the reversible one-electron reduction of nitrite to nitric oxide,
are members of a large family of multi-copper enzymes that require an interprotein electron transfer (ET) reaction with redox
partner proteins. Here, we show that the naturally fused type of CuNiR tethering a cytochrome c (Cyt c) at the C-terminus folds as a unique trimeric domain-swapped structure and has a self-sufficient electron flow system. The
C-terminal Cyt c domain is located at the surface of the type 1 copper (T1Cu) site in the N-terminal CuNiR domain from the adjacent subunit,
the heme-to-Cu distance (10.6 Å) of which is comparable to the transient ET complex of normal CuNiR with Cyt c. The structural aspects for the domain–domain interface and the ET kinetics indicate that the Cyt c–CuNiR domain interaction should be highly transient. The further electrochemical analysis of the interprotein ET reaction
with a cognate redox partner protein suggested that an electron is directly transferred from the partner to the T1Cu. Structural
and mechanistic comparisons of Cyt c–CuNiR with another cupredoxin-tethering CuNiR highlight the behaviours of extra domains on the fusion types of CuNiRs required
for ET through proteins.
Electron transfer reactions are essential for life because they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of ATP, and are involved in many reactions of intermediary metabolism. Key to these roles is the formation of transient inter-protein electron transfer complexes. The structural basis for the control of specificity between partner proteins is lacking because these weak transient complexes have remained largely intractable for crystallographic studies. Inter-protein electron transfer processes are central to all of the key steps of denitrification, an alternative form of respiration in which bacteria reduce nitrate or nitrite to N2 through the gaseous intermediates nitric oxide (NO) and nitrous oxide (N2O) when oxygen concentrations are limiting. The one-electron reduction of nitrite to NO, a precursor to N2O, is performed by either a haem- or copper-containing nitrite reductase (CuNiR) where they receive an electron from redox partner proteins a cupredoxin or a c-type cytochrome. Here we report the structures of the newly characterized three-domain haem-c-Cu nitrite reductase from Ralstonia pickettii (RpNiR) at 1.01 Å resolution and its M92A and P93A mutants. Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage. Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 (ref. 6), and mutagenesis studies provide direct evidence for the importance of a hydrogen-bonded water at the interface in electron transfer. The structure also provides an explanation for the preferential binding of nitrite to the reduced copper ion at the active site in RpNiR, in contrast to other CuNiRs where reductive inactivation occurs, preventing substrate binding.
Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.
Biological electron transfer is a fundamentally important reaction. Despite the apparent simplicity of these reactions (in that no bonds are made or broken), their experimental interrogation is often complicated because of adiabatic control exerted through associated chemical and conformational change. We have studied the nature of this control in several enzyme systems, cytochrome P450 reductase, methionine synthase reductase and copper-dependent nitrite reductase. Specifically, we review the evidence for conformational control in cytochrome P450 reductase and methionine synthase reductase and chemical control i.e. proton coupled electron transfer in nitrite reductase. This evidence has accrued through the use and integration of structural, spectroscopic and advanced kinetic methods. This integrated approach is shown to be powerful in dissecting control mechanisms for biological electron transfer and will likely find widespread application in the study of related biological redox systems.
The active site geometry of cytochrome (Cyt) c(551) and its mutated form containing Fe(II) and Fe(III) ions have been calculated using density functional theory (DFT)-based Becke's three-parameter hybrid exchange and Lee-Yang-Parr correlation (B3LYP) method. In addition, calculations have also been carried out using hybrid meta DFT-based M06 functional. The effect of the protein milieu on the active site geometry has also been probed using two-layer via our own N-layered integrated molecular orbital + molecular mechanics (ONIOM) method. Evidence from the calculations reveal that the active site geometry is not significantly affected by the oxidation state of metal ion. The difference in the geometry of the active site and that of the same with the entire protein environment is only minimal, which shows that the protein milieu does not influence the structure of the active site. The calculated electronic transition energies from the time-dependent DFT (TDDFT) calculations are in close agreement with the experimental values. Although there are no significant variations in the active site geometry upon oxidation, the changes in the electronic transition energies have been attributed to the reduction in the overlap of metal ion with the ligand orbitals. In addition, it is found that mutation does not influence the active site geometry and the electronic transition energies. Nevertheless, mutation leads to the formation of more compact structure than the native Cyt c(551).
Recent earth science studies have pointed out that massive acceleration of the global nitrogen cycle by anthropogenic addition of bio-available nitrogen has led to a host of environmental problems. Nitrous oxide (N(2)O) is a greenhouse gas that is an intermediate during the biological process known as denitrification. Copper-containing nitrite reductase (CuNIR) is a key enzyme in the process; it produces a precursor for N(2)O by catalysing the one-electron reduction of nitrite (NO2-) to nitric oxide (NO). The reduction step is performed by an efficient electron-transfer reaction with a redox-partner protein. However, details of the mechanism during the electron-transfer reaction are still unknown. Here we show the high-resolution crystal structure of the electron-transfer complex for CuNIR with its cognate cytochrome c as the electron donor. The hydrophobic electron-transfer path is formed at the docking interface by desolvation owing to close contact between the two proteins. Structural analysis of the interface highlights an essential role for the loop region with a hydrophobic patch for protein-protein recognition; it also shows how interface construction allows the variation in atomic components to achieve diverse biological electron transfers.
An open access copy of this article is available and complies with the copyright holder/publisher conditions. Nonrelativistic and relativistic Hartree-Fock (HF) and configuration interaction (CI) calculations have been performed in order to analyze the relativistic and correlation effects in various diatomic gold compounds. It is found that relativistic effects reverse the trend in most molecular properties down the group (11). The consequences for gold chemistry are described. Relativistic bond stabilizations or destabilizations are dependent on the electronegativity of the ligand, showing the largest bond destabilization for AuF (86 kJ/mol at the CI level) and the largest stabilization for AuLi (-174 kJ/mol). Relativistic bond contractions lie between 1.09 (AuH+) and 0.16 A (AuF). Relativistic effects of various other properties are discussed. A number of as yet unmeasured spectroscopic properties, such as bondlengths (re), dissociation energies (De), force constants (k e), and dipole moments (?e), are predicted.
The reduction of nitrite (NO2−) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this
study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel
steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope
effect (SKIE) of 1.3 ± 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation
experiments have been used to obtain single turnover data in H2O and D2O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE
of ∼1.33 ± 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substrate-free AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H2O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at
nitrite concentrations of >10 mm. As a consequence, the observed ET process was faster in D2O than in H2O above 0.1 mm nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum
value of ∼0.61 ± 0.02 between 3 and 10 mm. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound
AxNiR, and two protons were found to be involved in turnover.
In this study an attempt has been made to investigate the effect of metal ion substitution on the structure and spectra of azurin using two-layer ONIOM (our own N-layered integrated molecular orbital + molecular mechanics) approach. It is evident from the results that the overall structure of the protein is not altered by metal ion substitution; nevertheless, the metal ion binding site undergoes noticeable changes. The present study highlights the importance of protein milieu in the prediction of structure, electronic, and spectral properties of native and substituted azurins and illustrates the usefulness of ONIOM approach in the designing and engineering of metalloproteins.
Single-site catalysts offer great chances for unraveling the mechanism and the selectivity of catalytic mechanism, in particular when the system is experimentally well characterized. A particular interesting system of this type is the hydrogenation and the dimerization of ethene by the faujasite-supported complex [Rh(C2H4)2]+. We have examined this system computationally, treating periodic models with a density functional method. The complex [Rh(C2H4)2]+ binds in a bidentate fashion, as previously suggested, inside the faujasite supercage at the oxygen atoms of a 12-member ring. The calculations on this model complex showed ethene hydrogenation to be preferred over dimerization. The highest free energy barrier for forming a C–H bond was calculated at 33 kJ mol−1 at room temperature. This value is significantly lower than the lowest activation free energy, 97 kJ mol−1, calculated for C–C bond formation. The results of this mechanistic study allow one to rationalize the experimental observation that the faujasite-supported [Rh(C2H4)2]+ complex in the presence of H2 is active for hydrogenation, producing ethane as the main product.
As an essential component of amino acids and nucleic acids, N is a key element of life. In order for atmospheric (dinitrogen, N2) and environmental (nitrate and nitrite, NO3- and NO2 -) sources of N to be utilized in various forms of life, it must first be reduced to a more suitable form, such as ammonia (NH3). The Haber-Bosch process represents a major NH3 production process, which has greatly impacted the agricultural crop industry. This minireview discusses the recent electrochemistry of three key enzymes of the global biogeochemical N cycle (nitrogenase, nitrate reductase and nitrite reductase), in light of moving towards creating alternative NH3 production technologies.
The reaction mechanism of copper-containing nitrite reductase (CuNiR) has been proposed to include the two important events, the intramolecular electron transfer and the proton transfer. The two events have been suggested to be coupled, however, the order of these events is currently under debate. We investigated the entire enzyme reaction mechanism of the nitrite reduction on T2 Cu site in thermophilic Geobacillus CuNiR from Geobacillus thermodenitrificans NG80-2 (GtNiR) by using the DFT calculation method. We found significant conformational changes of His ligands coordinated to T2 Cu site on the nitrite binding during the catalytic reaction. The reduction potentials and pKa values calculated for the relevant protonation and reduction states show two possible routes, A and B. The reduction of T2 Cu site in the resting state is followed by the endothermic nitrite binding in route A, while the exothermic nitrite binding occurs prior to the reduction of Cu T2 site in route B. We concluded that our results support the random-sequential mechanism rather than the ordered-mechanism. .
Dissimilatory reduction of nitrite by copper-containing nitrite reductase (CuNiR) is an important step in geobiochemical nitrogen cycle. The proposed mechanisms for the reduction of nitrite by CuNiRs include the intramolecular electron and proton transfers, and these two events are understood to couple. Proton coupled electron transfer is one of the key process in enzyme reactions. We investigated the geometric structure of bound nitrite and the mechanism of nitrite reduction on CuNiR by using the density functional theory (DFT) calculations. Also, the proton transfer pathway, the key residues and their roles in reaction mechanism were clarified in this study. In our results, the reduction of T2 Cu site promotes the proton transfer and the hydrogen bond network around the binding site has an important role not only to stabilize the nitrite binding but also to promote the proton transfer to nitrite.
Using density functional theory (DFT), we studied denitrification reaction mechanisms of copper adducts of tris(pyrazolyl)methane and hydrotris(pyrazolyl)borate models of a copper nitrite reductase (Cu-NiR), and herein propose several possible reaction pathways including some parts that have never been examined previously. Since electron and proton transfer reactions participate in the enzymatic cycles of the Cu-NiR, and in the Gibbs energy of a proton in solution, G(H+) including the redox potential, Eredox, of the model Cu-NiR are also evaluated. Although the pathway, where a nitrite is provided as HNO2 is energetically preferable, and a well-known reaction pathway, passing through the resting state with an active site occupied by a water molecule where nitrite is provided as NO2- is the main recognized pathway under normal conditions. These features do not change whether the electron transfer occurs before a production of NO or not. However, our results suggest that the pathway involving HNO2 might become dominant at a low pH condition in conjunction with experimental results.
The solvation free energy of proton in methanol was calculated by B3LYP flavor of density functional calculations in combination with the Poisson-Boltzmann continuum solvation model. In order to check the adequacy of the computation level, the free energies of clustering in the gas phase were compared with the experimental data. The solvents were taken into account in a hybrid manner, i.e. one to five molecules of methanol were explicitly considered while other solvent molecules were represented with an implicit solvation model.
The Universal Force Field (UFF) (Rappe et al., J. Am. Chem. Soc. 1992) provides a general approach to molecular mechanics for molecules and materials composed of elements throughout the periodic table. Though the method is tunable by the specification of bond orders and the introduction of effective charges, the presently available list of atom types is insufficient to treat various systems containing transition metals, including metal organic frameworks (MOFs). As MOFs are composite materials built of a combination of individually stable building blocks, a plethora of MOF structures are possible, and the prediction of their structure with a low-cost method is important. We have extended the UFF parameter set to include transition metal elements Zn, Cu, Ni, Co, Fe, Mn, Cr, V, Ti, Sc, and Al, as they occur in MOFs, and have proposed additional O parameters that provide reliable structures of the metal oxide clusters of the connectors. We have benchmarked the performance of the MOF extension to UFF (UFF4MOF) with respect to experimentally available data and to DFT calculations. The parameters are available in various well-spread programs, including GULP, deMonNano, and ADF, and all information is provided to include them in other molecular mechanics codes.
A GUI (Graphic User Interface) for GAMESS / FMO (Fragment MO method) calculation was developed and implemented on Facio, the pre/post-processor for computational chemistry. By using this GUI, one can easily make FMO fragments for a given protein or nucleic acid and generate FMO input files.
Besides automatic fragmentation, one can manually define additional FMO fractioning points to non-peptide moiety of conjugated protein. For manual definition, a novel local structure viewer was also developed. The desired local structure is defined by an atom number or by a fragment number. Using this local structure viewer, one can check the structural validity of each FMO fragment and perform manual definition of additional fractioning points on the fragment. Manually defined fractioning points can be saved as "fragment definition file", which can be loaded later to set the predefined fractioning points.
For the optimization of hydrogen atoms automatically added to the PDB file of a protein or nucleic acid which lacks hydrogen atoms, a fragment optimization function was implemented for the GUI. While a molecular dynamics method or molecular mechanics method is usually used for this purpose, the new optimization method utilizes a molecular orbital method, such as PM3 or STO-3G of GAMESS.
Oxidation of uracil (U) and thymine (5-Me-U) are believed to play a role in genetic instability because of the changes these oxidations cause in the ionization constants (pK(a) values), which in turn affects the base pairing and hence coding. However, interpretation of the experimental evidence for the changes of pK(a) with substitution at LT has been complicated by the presence of two sites (N1 and N3) for ionization. We show that a procedure using first principles quantum mechanics (density functional theory with generalized gradient approximation, B3LYP, in combination with the Poisson-Boltzmann continuum-solvation model) predicts such pK(a) values for a series of 5-substituted uracil derivatives in excellent correlation with experiment. In particular, this successfully resolves which cases prefer ionization at N1 and N3. Such first principles predictions of ionization constant should be useful for predicting and interpreting pK(a) for other systems.
The one-electron addition on the disulfide bond of thioredoxin was studied by a QM/MM procedure. Three methodological aspects were considered: the presence of a MM surrounding, the choice of the QM method and the QM/MM partitioning. We show that the environment has a relatively small effect on geometry but it strongly influences electronic affinity (EA). Even with the MM part, B3LYP and HF methods are still inadequate and at least MP2 is needed for the treatment of the (2c–3e) bond. However, a relatively restrained QM part seems to be sufficient for modelling this electronic property.
Nonrelativistic and quasirelativistic ab‐initio pseudopotentials representing the Ne‐like X(Z−10)+ cores (X=Sc–Zn) of the first row transition metals and optimized (8s7p6d1f )/[6s5p3d1f ]‐GTO valence basis sets for use in molecular calculations have been generated. Excitation and ionization energies of the low lying states of Sc through Zn from numerical HF‐ as well as SCF‐ and CI(SD)‐pseudopotential calculations using the derived basis sets differ by less than 0.1 eV from corresponding all‐electron results.
The value of the proton hydration free energy, ΔGhyd(H+), has been quoted in the literature to be from −252.6 to −262.5 kcal/mol. In this article, we present a theoretical model for calculating the hydration free energy of ions in aqueous solvent and use this model to calculate the proton hydration free energy, ΔGhyd(H+), in an effort to resolve the uncertainty concerning its exact value. In the model we define ΔGhyd(H+) as the free energy change associated with the following process: ΔG[H+(gas)+H2nOn(aq)→H+(H2nOn)(aq)], where the solvent is represented by a neutral n-water cluster embedded in a dielectric continuum and the solvated proton is represented by a protonated n-water cluster also in the continuum. All solvated species are treated as quantum mechanical solutes coupled to a dielectric continuum using a self consistent reaction field cycle. We investigated the behavior of ΔGhyd(H+) as the number of explicit waters of hydration is increased from n = 1 to n = 6. As n increases from 1 to 3, the hydration free energy decreases dramatically. However, for n = 4–6 the hydration free energy maintains a relatively constant value of −262.23 kcal/mol. These results indicate that the first hydration shell of the proton is composed of at least four water molecules. The constant value of the hydration free energy for n ≥ 4 strongly suggests that the proton hydration free energy is at the far lower end of the range of values that have been proposed in the literature. © 1998 American Institute of Physics.
A method is presented to determine the absolute hydration enthalpy of the proton, ΔHaq°[H+], from a set of cluster-ion solvation data without the use of extra thermodynamic assumptions. The absolute proton hydration enthalpy has been found to be 50 kJ/mol different than traditional values and has been more precisely determined (by about an order of magnitude). Conventional ion solvation properties, based on the standard heat of formation of H+(aq) set to zero, have been devised that may be confusing to the uninitiated but are useful in thermochemical evaluations because they avoid the unnecessary introduction of the larger uncertainties in our knowledge of absolute values. In a similar strategy, we have motivated the need for a reassessment of ΔHaq°[H+] by the trends with increased clustering in conventional cluster-ion solvation enthalpy differences for pairs of oppositely charged cluster ions. The consequences of particular preferred values for ΔHaq°[H+] may be evaluated with regard to cluster-ion properties and how they connect to the bulk. While this approach defines the problem and is strongly suggestive of the currently determined proton value, it requires extra thermodynamic assumptions for a definitive determination. Instead, a unique reassessment has been accomplished without extra thermodynamic assumptions, based on the known fraction of bulk absolute solvation enthalpies obtained by pairs of oppositely charged cluster ions at particular cluster sizes. This approach, called the cluster-pair-based approximation for ΔHaq°[H+], becomes exact for the idealized pair of ions that have obtained the same fraction of their bulk values at the same cluster size. The true value of ΔHaq°[H+] is revealed by the linear deviations of real pairs of ions from this idealized behavior. Since the approximation becomes exact for a specific pair of oppositely charged ions, the true value of ΔHaq°[H+] is expected to be commonly shared on plots of the approximation vs the difference in cluster-ion solvation enthalpy for pairs of ions sharing the same number of solvating waters. The common points on such plots determine values of −1150.1 ± 0.9 kJ/mol (esd) for ΔHaq°[H+] and −1104.5 ± 0.3 kJ/mol (esd) for ΔGaq°[H+]. The uncertainties (representing only the random errors of the procedure) are smaller than expected because the cluster data of 20 different pairings of oppositely charged ions are folded into the determination.
The dielectric constants of myoglobin, apomyoglobin, the B fragment of staphylococcal protein A, and the immunoglobulin-binding domain of streptococcal protein G are calculated from 1−2 ns molecular dynamics simulations in water, using the Fröhlich−Kirkwood theory of dielectrics. This dielectric constant is a direct measure of the polarizability of the protein medium and is the appropriate macroscopic quantity to measure its relaxation properties in response to a charged perturbation, such as electron transfer, photoexcitation, or ion binding. In each case the dielectric constant is low (2−3) in the protein interior, then rises to 11−21 for the whole molecule. The large overall dielectric constant is almost entirely due to the charged protein side chains, located at the protein surface, which have significant flexibility. If these are viewed instead as part of the outer solvent medium, then the remainder of the protein has a low dielectric constant of 3−6 (depending on the protein), comparable to that of dry protein powders. Similar results were already observed for ferro- and ferricytochrome c, and are probably valid for many or most stable globular proteins in solution, leading to a rather comprehensive picture of charge screening and the dielectric constant of proteins. This picture suggests ways, and supports some ongoing efforts, to improve current Poisson−Boltzmann models. Indeed, treating a protein as a homogeneous, low dielectric medium is likely to underestimate the actual dielectric relaxation of the protein; this would affect calculations of the self-energy of titrating protons, or the reorganization energy of a redox electron.
A detailed thermodynamic analysis of principles involved in the determination of absolute half-cell potentials is presented. An absolute half-cell potential cannot be obtained by thermodynamic (equilibrium) measurements alone, and probably not by measurement alone even if nonequilibrium measurements are admitted. Some residual theoretical assessment is always necessary usually involving an interfacial dipole layer. However, one does have the option of choosing the residual dipole problem by selecting the overall method. We select a method which leaves a dipole problem at a solid interface where theory may hopefully be applied most expeditiously. Our value for the absolute SHE potential is -4.43 V. With it we are now in possession of five potentials which agree fairly closely, although each was derived by a totally different method.
Solvation free energies and pK(a) values of models for ionizable side chains of amino acids are calculated by using continuum dielectric methods; integral equation techniques are also investigated. The dependence of the solvation free energies on the parameters (charges and van der Waals interactions used to describe the model compounds) is explored by comparing different sets that are being used in protein and liquid simulations. The solvation free energies calculated with both continuum and integral equation methods and various parameter sets agree qualitatively with experiment but are not accurate enough to yield absolute pK(a) values. To obtain the experimental solvation free energies and pK(a) values of the model compounds with the continuum dielectric method, an adjusted parameter set is introduced; only very small changes from the standard parameter values are required. The set of calibrated parameters is tested on some bifunctional compounds and yields pK(a) changes in reasonable agreement with experiment. However, the pK(a) changes are very sensitive to the solution conformation. This may result in large pK(a) errors if conformational changes (e.g., between the ionized and neutral species) are not taken into account.
A new molecular mechanics force field, the Universal force field (UFF), is described wherein the force field parameters are estimated using general rules based only on the element, its hybridization, and its connectivity. The force field functional forms, parameters, and generating formulas for the full periodic table are presented.
We report here an approach for predicting charge distributions in molecules for use in molecular dynamics simulations. The input data are experimental atomic ionization potentials, electron affinities, and atomic radii. An atomic chemical potential is constructed by using these quantities plus shielded electrostatic interactions between all charges. Requiring equal chemical potentials leads to equilibrium charges that depend upon geometry. This charge equilibrium (QEq) approach leads to charges in excellent agreement with experimental dipole moments and with the atomic charges obtained from the electrostatic potentials of accurate ab initio calculations. QEq can be used to predict charges for any polymer, ceramic, semiconductor, or biological system, allowing extension of molecular dynamics studies to broad classes of new systems. The charges depend upon environment and change during molecular dynamics calculations. We indicate how this approach can also be used to predict infrared intensities, dielectric constants, and other charge-related properties.
The possible orientation of non-aqueous solvent molecules at the free surface and the metal/liquid interface is discussed by analysing existing data of Volta potential difference for Hg/non-aqueous solvent contacts and by plotting potentials of zero charge against the work functions for different metals. The estimates are discussed in the light of different sources of experimental evidence. The need for a solvent-independent potential scale is emphasized and the difference is shown between the absolute potential scale in different solvents and the scale normalized to the hydrogen electrode in water. The temperature coefficients of the potential of zero charge of Hg are examined after correction for the temperature coefficient of the work function of the metal, and the relation of their sign to the orientation of the solvent at the Hg/solution interface is discussed. The factors responsible for the interfacial orientation of solvent molecules are analysed and the specific role of the metal surface is tentatively outlined. Finally, the interfacial orientation in mixed solvents is briefly discussed.
Native nitrite reductases (NIRs) containing both type 1 and 2 Cu ions and type 2 Cu-depleted (T2D) NIRs from three denitrifying
bacteria (Achromobacter cycloclastes IAM 1013, Alcaligenes xylosoxidans NCIB 11015, and Alcaligenes xylosoxidans GIFU 1051) have been characterized by electronic absorption, circular dichroism, and electron paramagnetic resonance spectra.
The characteristic visible absorption spectra of these NIRs are due to the type 1 Cu centers, while the type 2 Cu centers
hardly contribute in the same region. The intramolecular electron transfer (ET) process from the type 1 Cu to the type 2 Cu
in native NIRs has been observed as the reoxidation of the type 1 Cu(I) center by pulse radiolysis, whereas no type 1 Cu in
T2D NIRs exhibits the same reoxidation. The ET process obeys first-order kinetics, and observed rate constants are 1400–1900
s–1 (t1/2 = ca. 0.5 ms) at pH 7.0. In the presence of nitrite, the ET process also obeys first-order kinetics, with rate constants
decreased by factors of 1/12–1/2 at the same pH. The redox potential of the type 2 Cu site is estimated to be +0.24 - +0.28 V,
close to that of the type 1 Cu site. Nitrate and azide ions bound to the type 2 Cu site change the redox potential. Nitrite
also would shift the redox potential of the type 2 Cu by coordination, and hence the intramolecular ET rate constant is decreased.
Pulse radiolysis experiments on T2D NIRs in the presence of nitrite demonstrate that the type 1 Cu(I) site is slowly oxidized
with a first-order rate constant of 0.03 s–1 at pH 7.0, suggesting that nitrite bound to the protein accepts an electron from the type 1 Cu. This result is in accord
with the finding that T2D NIRs show enzymatic activities, although they are lower than those of the native enzymes.
Dissimilatory nitrite reductase (NIR) is a key enzyme in the anaerobic respiratory pathway of denitrifying bacteria. There are two types of NIR, one of which contains copper and the other heme. Cu-containing NIR (Cu NIR) has the trimeric structure with one type 1 Cu (blue copper) atom and one type 2 Cu (nonblue copper) atom in each subunit. The type 1 Cu atom bound by 2His, Cys, and Met accepts one electron from an electron donor protein and shows an intense color, blue or green. The type 2 Cu atom bound by 3His and a solvent (H2O or OH−) is a reduction center of nitrite to NO. The intramolecular long-range electron transfer process is observed from the type 1 site to the type 2 Cu site with a half-life period of ca. 0.3 ms. The present review deals with (i) spectroscopic characterization of Cu NIR’s, (ii) structures of Cu NIR’s, and (iii) functions of Cu NIR’s (intermolecular electron transfer process, intramolecular electron transfer process, and reduction of nitrite ion).
Cytochrome c nitrite reductase catalyzes the six-electron, seven-proton reduction of nitrite to ammonia without release of any detectable reaction intermediate. This implies a unique flexibility of the active site combined with a finely tuned proton and electron delivery system. In the present work, we employed density functional theory to study the recharging of the active site with protons and electrons through the series of reaction intermediates based on nitrogen monoxide [Fe(II)-NO(+), Fe(II)-NO·, Fe(II)-NO(-), and Fe(II)-HNO]. The activation barriers for the various proton and electron transfer steps were estimated in the framework of Marcus theory. Using the barriers obtained, we simulated the kinetics of the reduction process. We found that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers (PCETs) or in the form of three consecutive elementary steps involving reduction, PCET, and protonation. Kinetic simulations revealed the recharging through two PCETs to be a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO· intermediate. The radical transfer role for the active-site Tyr(218), as proposed in the literature, cannot be confirmed on the basis of our calculations. The role of the highly conserved calcium located in the direct proximity of the active site in proton delivery has also been studied. It was found to play an important role in the substrate conversion through the facilitation of the proton transfer steps.
The purpose of this paper is 2-fold. First, we present several extensions to the ONIOM(QM: MM) scheme. In its original formulation, the electrostatic interaction between the regions is included at the classical level. Here we present the extension to electronic embedding. We show how the behavior of ONIOM with electronic embedding can be more stable than QM/ MM with electronic embedding. We also investigate the link atom correction, which is implicit in ONIOM but not in QM/ MM. Second, we demonstrate some of the practical aspects of ONIOM( QM: MM) calculations. Specifically, we show that the potential surface can be discontinuous when there is bond breaking and forming closer than three bonds from the MM region.
Recently, studies have been reported in which fluorescently labeled redox proteins have been studied with a combination of spectroscopy and electrochemistry. In order to understand the effect of the dye on the protein-electrode interaction, voltammetry and surface analysis have been performed on protein films of dye-labeled and unlabeled forms of a cysteine-surface variant (L93C) and the wild type (wt) of the copper containing nitrite reductase (NiR) from Alcaligenes faecalis S6. The protein has been adsorbed onto gold electrodes modified with self-assembled monolayers (SAMs) made up of 6-mercaptohexanol (6-OH) and mixtures of various octanethiols. Electrochemical and surface-analytical techniques were utilized to explore the influence of the SAM composition on wt and L93C NiR enzyme activity and the orientation of the enzyme molecules with respect to the electrode/SAM. The unlabeled L93C NiR enzyme is only electroactive on mixed SAMs composed of positive 8-aminooctanethiol (8-NH(2)) and 8-mercaptooctanol (8-OH). No enzymatic activity is observed on SAMs consisting of pure 6-OH, 8-OH, or pure 8-NH(2). Modification of L93C NiR with the ATTO 565 dye resulted in enzymatic activity on SAMs of 6-OH, but not on SAMs of 8-OH. Quartz crystal microbalance with dissipation measurements show that well-ordered and rigid protein films (single orientation of the protein) are formed when NiR is electroactive. By contrast, electrode-NiR combinations for which no electrochemical activity is observed still have NiR adsorbed on the surfaces, but a less-structured and water-rich film is formed. For the unlabeled L93C NiR, bilayer formation is observed, suggesting that the Cys93 residue is orientated away from the surface and able to form disulfide bridges to a second layer of L93C NiR. The results indicate that interfacial electron transfer is only possible if the negatively charged surface patch surrounding the electron-entry site of NiR is directed toward the electrode. This can be achieved either by introducing positive charges in the SAM or, when the SAM does not carry a charge, by labeling the enzyme with an ATTO 565 dye, which has some hydrophobic character, close to the electron entry site of the NiR.
The reduction of nitrite to nitric oxide in dissimilatory denitrification is carried out by copper nitrite reductases (CuNIRs) via a type 2 copper site. Extended studies on CuNIRs in combination with model complexes have allowed for the establishment of two potential mechanisms for this transformation. Recent experimental and computational results have revealed further details of this process. In addition, the interaction of NO with copper sites has recently gained much attention. This review discusses recent results in the context of the known coordination chemistry of CuNIRs.
A combined fluorescence and electrochemical method is described that is used to simultaneously monitor the type-1 copper oxidation state and the nitrite turnover rate of a nitrite reductase (NiR) from Alcaligenes faecalis S-6. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labeled enzyme molecules immobilized on modified gold electrodes, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by Förster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. The homotrimeric structure of the enzyme is reflected in heterogeneous interfacial electron-transfer kinetics with two monomers having a 25-fold slower kinetics than the third monomer. The intramolecular electron-transfer rate between the type-1 and type-2 copper site changes at high nitrite concentration (≥520 μM), resulting in an inhibition effect at low pH and a catalytic gain in enzyme activity at high pH. We propose that the intramolecular rate is significantly reduced in turnover conditions compared to the enzyme at rest, with an exception at low pH/nitrite conditions. This effect is attributed to slower reduction rate of type-2 copper center due to a rate-limiting protonation step of residues in the enzyme's active site, gating the intramolecular electron transfer.
We demonstrated recently that two protons are involved in reduction of nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent nitrite reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S--H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ~70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, in the N90S and N90S--H254F variants, where the T1Cu site redox potential is elevated by ∼60 mV, inter-Cu ET is only observed in the presence of nitrite. From these results it is evident that the Asn90 channel is the main proton channel in AxNiR, though protons can still reach the active site if this channel is disrupted. Crystallographic structures provide a clear structural rationale for these observations, including restoration of the proton delivery via a significant movement of the loop connecting the T1Cu ligands Cys130 and His139 that occurs on binding of nitrite. Notably, a role for this loop in facilitating interaction of cytochrome c(551) with Cu NiR has been suggested previously based on a crystal structure of the binary complex.
Cytochrome c nitrite reductase is a homodimeric enzyme, containing five covalently attached c-type hemes per subunit. Four of the heme irons are bishistidine-ligated, whereas the fifth, the active site of the protein, has an unusual lysine coordination and calcium site nearby. A fascinating feature of this enzyme is that the full six-electron reduction of the nitrite is achieved without release of any detectable reaction intermediate. Moreover, the enzyme is known to work over a wide pH range. Both findings suggest a unique flexibility of the active site in the complicated six-electron, seven-proton reduction process. In the present work, we employed density functional theory to study the energetics and kinetics of the initial stages of nitrite reduction. The possible role of second-sphere active-site amino acids as proton donors was investigated by taking different possible protonation states and geometrical conformations into account. It was found that the most probable proton donor is His(277), whose spatial orientation and fine-tuned acidity lead to energetically feasible, low-barrier protonation reactions. However, substrate protonation may also be accomplished by Arg(114). The calculated barriers for this pathway are only slightly higher than the experimentally determined value of 15.2 kcal/mol for the rate-limiting step. Hence, having proton-donating side chains of different acidity within the active site may increase the operational pH range of the enzyme. Interestingly, Tyr(218), which was proposed to play an important role in the overall mechanism, appears not to take part in the reaction during the initial stage.
The capabilities and limitations of the Becke-3-Lee-Yang-Parr (B3LYP) density functional theory (DFT) for modeling proton coupled electron transfer (PCET) in the mixed-valence oxomanganese complex 1 [(bpy)(2)Mn(III)(mu-O)(2)Mn(IV)(bpy)(2)](3+) (bpy = 2,2'-bipyridyl) are analyzed. Complex 1 serves as a prototypical synthetic model for studies of redox processes analogous to those responsible for water oxidation in the oxygen-evolving complex (OEC) of photosystem II (PSII). DFT B3LYP free energy calculations of redox potentials and pKa's are obtained according to the thermodynamic cycle formalism applied in conjunction with a continuum solvation model. We find that the pKa's of the oxo-ligands depend strongly on the oxidation states of the complex, changing by approximately 10 pH units (i.e., from pH~2 to pH~12) upon III,IV-->III,III reduction of complex 1. These computational results are consistent with the experimental pKa's determined by solution magnetic susceptibility and near-IR spectroscopy as well as with the pH dependence of the redox potential reported by cyclic voltammogram measurements, suggesting that the III,IV-->III,III reduction of complex 1 is coupled to protonation of the di-mu-oxo bridge as follows: [(bpy)(2)Mn(III)(mu-O)(2) Mn(IV)(bpy)(2)](3+)+H(+)+e(-)-->[(bpy)(2)Mn(III)(mu-O)(mu-OH)Mn(III)(bpy)(2)](3+). It is thus natural to expect that analogous redox processes might strongly modulate the pKa's of oxo and hydroxo/water ligands in the OEC of PSII, leading to deprotonation of the OEC upon oxidation state transitions.
[Fe-Fe]-hydrogenases are enzymes that reversibly catalyze the reaction of protons and electrons to molecular hydrogen, which occurs in anaerobic media. In living systems, [Fe-Fe]-hydrogenases are mostly used for H(2) production. The [Fe-Fe]-hydrogenase H-cluster is the active site, which contains two iron atoms. The latest theoretical investigations1,2 advocate that the structure of di-iron air inhibited species are either Fe(p) (II)-Fe(d) (II)-O-H(-), or Fe(p) (II)-Fe(d) (II)-O-O-H, thus O(2) has to be prevented from binding to Fe(d) in all di-iron subcluster oxidation states in order to retain a catalytically active enzyme. By performing residue mutations on [Fe-Fe]-hydrogenases, we were able to weaken O(2) binding to distal iron (Fe(d)) of Desulfovibrio desulfuricans hydrogenase (DdH). Individual residue deletions were carried out in the 8 A apoenzyme layer radial outward from Fe(d) to determine what residue substitutions should be made to weaken O(2) binding. Residue deletions and substitutions were performed for three di-iron subcluster oxidation states, Fe(p) (II)-Fe(d) (II), Fe(p) (II)-Fe(d) (I), and Fe(p) (I)-Fe(d) (I) of [Fe-Fe]-hydrogenase. Two deletions (DeltaThr(152) and DeltaSer(202)) were found most effective in weakening O(2) binding to Fe(d) in Fe(p) (II)-Fe(d) (I) hydrogenase (DeltaG(QM/MM) = +5.4 kcal/mol). An increase in Gibbs' energy (+2.2 kcal/mol and +4.4 kcal/mol) has also been found for Fe(p) (II)-Fe(d) (II), and Fe(p) (I)-Fe(d) (I) hydrogenase respectively. pi-backdonation considerations for frontier molecular orbital and geometrical analysis corroborate the Gibbs's energy results.
A theoretical investigation on the interaction of myristic fatty acid (M) with Acutohaemolysin and Piratoxin-II of PLA2 family is performed using two layered ONIOM (B3LYP/6-31G*: UFF) method. The results predict that though proteins show revulsion to incoming fatty acid, the interaction of the phenyl ring of Phenylalanine restricts the passage of M through the channel. To unveil the nature of interaction of M, quantum chemical studies are carried out on the palindromic tripeptides Alanine-Phenylalanine-Alanine (AFA) and Alanine-Valine-Alanine (AVA) present in Acutohaemolysin and Piratoxin-II at B3LYP/6-311G** level of theory. The mode of interaction of the fatty acid with protein is electrostatic, confirmed further through molecular electrostatic potential (MEP) maps. The AFA shows stronger interaction than AVA, validating the impact of mutation on catalytic activity. Further such strong interaction and hence the higher probability of prohibition for catalytic activity exists only when the fatty acid interacts at the center of phenyl ring than at its edges. The preferred secondary structural configuration and conformational properties of AVA and AFA also validate the strong interaction of fatty acid with Phenylalanine. In general, this theoretical investigation shows that the loss of catalytic activity would take place only when fatty acid interacts at the center of phenyl ring.
Proton and electron delivery to the catalytic site and their associated pathways are crucial elements in understanding the mechanisms of redox enzymes. Two distinct proton channels have previously been identified in copper nitrite reductases based on high- to atomic-resolution crystal structures. These were assigned as the "primary" and "high-pH" proton channels and link the catalytic type 2 Cu center to the enzyme surface. Residue His254 has been identified as a key residue in the primary proton channel from the catalytic T2Cu site to the surface, while Asn90 is thought to be a key residue in the high-pH channel. The structure of the His254Phe mutant was previously determined to 1.85 A resolution, revealing disruption in the H-bonding network of the primary proton channel. The effect of the mutation on proton transfer was not established as the T2Cu center was unusually occupied by Zn. New growth protocols have now led to the incorporation of copper at this site, and here we present spectroscopic, catalytic activity, and structural data for the Cu-loaded H254F mutant of AxNiR. Surprisingly, this species exhibits essentially full catalytic activity, despite the clear disruption of the primary proton channel. In contrast, the Asn90Ser mutation disrupts H-bonding in the high-pH proton channel and results in an approximately 70% decrease in specific activity. These mutations do not change the apparent K(m) for nitrite, and thus, these data clearly demonstrate a role for the high-pH proton channel in the delivery of protons to the catalytic T2Cu center at physiological pH values; it may in fact be the main source of protons to the T2Cu center.
A combination of spectroscopy and DFT calculations has been used to define the geometric and electronic structure of the nitrite bound type 2 (T2) copper site at high and low pH in nitrite reductase from Rhodobacter sphaeroides. At high pH there is no electron transfer from reduced type 1 (T1) to the nitrite bound T2 copper, while protonation triggers T1 --> T2 electron transfer and generation of NO. The DFT calculated reaction coordinate for the N-O bond cleavage in nitrite reduction by the reduced T2 copper suggests that the process is best described as proton transfer triggering electron transfer. Bidentate nitrite binding to copper is calculated to play a major role in activating the reductive cleavage of the nitrite bond through backbonding combined with stabilization of the (-)OH product by coordination to the Cu(2+).