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Calculated vibrational modes for select low-frequency vibrations in [(NH3)MoO(qdt)] 1+. The images overlay the displacements of the atoms in the model at the extrema of their vibrational motion. The calculated frequencies for this model are 352 (ν′ 1), 396 (ν′6), and 385 (ν′3) cm-1 .
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![Figure 3. Basic stereochemistry of the [(Tp*)Mo V O(dithiolene)] system...](profile/Frank-Inscore/publication/7332779/figure/fig2/AS:739635265282048@1553354197455/Basic-stereochemistry-of-the-TpMo-V-Odithiolene-system-showing-the-bidentate_Q320.jpg)
X-ray crystallography and resonance Raman (rR) spectroscopy have been used to further characterize (Tp*)MoO(qdt) (Tp* is hydrotris(3,5-dimethyl-1-pyrazolyl)borate and qdt is 2,3-quinoxalinedithiolene), which represents an important benchmark oxomolybdenum mono-dithiolene model system relevant to various pyranopterin Mo enzyme active sites, includin...
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... have investigated the potential for vibrational mode mixing using a vibrational frequency analysis within the Gaussian03 suite of programs in order to probe the nature of the vibrational normal modes in [(NH 3 ) 3 MoO(qdt)] 1+ , a computational model for 4 where the tris-pyrazolylborate ligand has been effectively modeled by three coordinated amine donors. It is clear that extensive mode mixing occurs and the true vibrational normal modes may best be described as symmetry-adapted linear combinations of the original ν 1 and ν 6 symmetry coordinates, and these are shown in Figure ( 8. The calculated frequencies for this model are 352 (ν′ 1 ), 385 (ν′ 3 ), and 396 cm -1 (ν′ 6 ) ( Figure 9). These calculated frequencies are in excellent agreement with the experimental data and allow us to assign the 348 and 407 cm -1 modes of 4 as ν′ 1 and ν′ 6 , respectively. ...
Citations
... MCD spectroscopy has been used to probe the electronic structure of chicken sulfite oxidase that was poised in the catalytically relevant [Mo(V):Fe(II)] state [145,147] ( Figure 6, middle). No charge transfer transitions were observed at energies below~17,000 cm −1 and this was interpreted to result from a reduction in S ip -S op orbital mixing and S op -Mo d(xy) covalency, in addition to a small dithiolene fold angle [130,145,[147][148][149][150][151]. A band at 22,250 cm −1 was observed as a positive C-term and assigned as a S σ (cysteine)→Mo(xy) LMCT transition. ...
A concise review is provided of the contributions that various spectroscopic methods have made to our understanding of the physical and electronic structures of mononuclear molybdenum enzymes. Contributions to our understanding of the structure and function of each of the major families of these enzymes is considered, providing a perspective on how spectroscopy has impacted the field.
... Perhaps the most characterized benchmark oxomolybdenum dithiolene complexes are Tp*MoO(bdt) [88], Tp*MoO(tdt) [88], and Tp*MoO(qdt) [89] (Tp* = hydrotris-(3,5-dimethyl-1-pyrazolyl)borate; bdt = 1,2-benzenedithiolate; tdt = 3,4-toluenedithiolate; qdt = quinoxaline dithiolate). These paramagnetic d 1 Mo(V) complexes are important for understanding the electronic structure of molybdoenzymes that possess a single terminal Mo ---O bond oriented cis to a dithiolene chelate, since this coordination geometry is encountered in many pyranopterin containing Mo enzyme forms [1][2][3]6,17,19,88,90]. ...
... The resonance Raman results for Tp*MoO(qdt) were similar to those of Tp*MoO(bdt), displaying vibrational bands at 348 and 407 cm − 1 . However, a computational analysis of the normal modes showed that the S-Mo-S (ν Mo-S ) stretch and bend (δ S-Mo-S ) vibrations were mixed, leading to a different normal mode description for the MoOS 2 core [89]. A key observation from these model studies is the small number of vibrational modes in the low-frequency (~200-1000 cm − 1 ) region of the Raman spectra, which is in stark contrast to what is observed in many of the enzymes, where low-frequency Mo -S vibrations can be coupled with other low-frequency modes of the pyranopterin or even kinematically coupled with other protein modes. ...
... They showed that the C--C stretching frequency is downshifted by ~7 cm − 1 to 1568 cm − 1 for DMSOR red , consistent with a change in Mo-dithiolene covalency as a function of the Mo ion oxidation state. The dithiolene C--C stretching vibration is expected to be observed in the 1300-1600 cm − 1 region of the Raman spectrum [100,105] with frequencies that are anticipated to vary as a function of the oxidation state of the dithiolene [12,13,106,107], the oxidation state of the metal, and the magnitude of the "sulfur-fold" distortion within the five-membered chelate ring [18,89,106]. In 1995 Spiro and coworkers noted that the 1568 cm − 1 band that had previously been observed for DMSOR red , and assigned as a C--C stretching vibration, was likely an experimental artifact [108]. ...
Resonance Raman spectroscopy (rR) is a powerful spectroscopic probe that is widely used for studying the geometric and electronic structure of metalloproteins. In this focused review, we detail how resonance Raman spectroscopy has contributed to a greater understanding of electronic structure, geometric structure, and the reaction mechanisms of pyranopterin molybdenum enzymes. The review focuses on the enzymes sulfite oxidase (SO), dimethyl sulfoxide reductase (DMSOR), xanthine oxidase (XO), and carbon monoxide dehydrogenase. Specifically, we highlight how Mo-Ooxo, Mo-Ssulfido, Mo-Sdithiolene, and dithiolene CC vibrational modes, isotope and heavy atom perturbations, resonance enhancement, and associated Raman studies of small molecule analogs have provided detailed insight into the nature of these metalloenzyme active sites.
... Such MPT hydrogen bonding networks may also control the degree of MPT non-planarity and the magnitude of the MoS 2 C 2 chelate ring fold angle [21]. This would allow for static and dynamic Mo-dithiolene charge redistribution, also known as the electronic buffer effect [100], along the electron and atom transfer reaction coordinates during catalysis [101]. In this way, enzyme-MPT hydrogen bonding would provide a means of converting protein vibrational energy into specific electronic structure changes at the Mo center to modulate metal-dithiolene covalency and the metal ion reduction potential [4,[102][103][104][105][106]. ...
The last 20 years have seen a dramatic increase in our mechanistic understanding of the reactions catalyzed by pyranopterin Mo and W enzymes. These enzymes possess a unique cofactor (Moco) that contains a novel ligand in bioinorganic chemistry, the pyranopterin ene-1,2-dithiolate. A synopsis of Moco biosynthesis and structure is presented, along with our current understanding of the role Moco plays in enzymatic catalysis. Oxygen atom transfer (OAT) reactivity is discussed in terms of breaking strong metal-oxo bonds and the mechanism of OAT catalyzed by enzymes of the sulfite oxidase (SO) family that possess dioxo Mo(VI) active sites. OAT reactivity is also discussed in members of the dimethyl sulfoxide (DMSO) reductase family, which possess des-oxo Mo(IV) sites. Finally, we reveal what is known about hydride transfer reactivity in xanthine oxidase (XO) family enzymes and the formate dehydrogenases. The formal hydride transfer reactivity catalyzed by xanthine oxidase family enzymes is complex and cleaves substrate C-H bonds using a mechanism that is distinct from monooxygenases. The chapter primarily highlights developments in the field that have occurred since ~2000, which have contributed to our collective structural and mechanistic understanding of the three canonical pyranopterin Mo enzymes families: XO, SO, and DMSO reductase.
... Accurate fold angles are difficult to determine for large protein molecules, but values ranging from 6-33° have been calculated for various molybdenum enzymes [31]. The binding of substrate or inhibitors, and/or dynamic conformational changes in the protein, are expected to modulate the active site chelate fold angle and thereby affect enzyme reactivity [4,32]. ...
... Experimental investigation of the electronic structures of the Mo centers of enzymes is difficult because of the intense absorptions from other chromophores (e.g., the b-type heme in sulfite oxidase and iron sulfur centers and FAD in xanthine oxidase) [37][38][39][40][41]. However, the effects of dithiolene coordination on electronic structure have been investigated for model oxo-Mo(V) compounds ( Figure 6) by electronic absorption, XAS, magnetic circular dichroism (MCD), and resonance Raman (rR) spectroscopies [12,[14][15][16][17]32,33,[42][43][44][45][46][47][48][49][50][51]. For Tp*MoO(bdt), the electronic absorptions at 19,400 cm −1 (Band 4) and 22,100 cm −1 (Band 5) are assigned to S ➝ Mo charge transfer bands ( Figure 7A) [12]. ...
... Thus, the instantaneous generation of a hole on the Mo center (Mo(IV)-P 0 → Mo(V)-P•) by photoexcitation is felt by the dithiolene chelate and extends all the way to the amino terminus of the PDT. The most resonantly enhanced mode in this spectral region is Band C, the symmetric S-Mo-S dithiolene core stretching, and the frequency of this mode and Band D are similar to those observed in Tp*MoO(bdt) [12,32], which were assigned as the chelate ring symmetric S-Mo-S stretching and bending vibrations, respectively. Band A is significant, since it possesses dithiolene ring folding character indicating that electron density changes at Mo are buffered by a distortion along this low-frequency coordinate, as has been observed in the various model systems described in this review. ...
Here we highlight past work on metal–dithiolene interactions and how the unique electronic structure of the metal–dithiolene unit contributes to both the oxidative and reductive half reactions in pyranopterin molybdenum and tungsten enzymes. The metallodithiolene electronic structures detailed here were interrogated using multiple ground and excited state spectroscopic probes on the enzymes and their small molecule analogs. The spectroscopic results have been interpreted in the context of bonding and spectroscopic calculations, and the pseudo-Jahn–Teller effect. The dithiolene is a unique ligand with respect to its redox active nature, electronic synergy with the pyranopterin component of the molybdenum cofactor, and the ability to undergo chelate ring distortions that control covalency, reduction potential, and reactivity in pyranopterin molybdenum and tungsten enzymes.
... Figures 5 and 6 show the structures of the anionic complexes with line drawings to aid interpretation. Complexes 2 and 4 are structurally similar to other Tp*−Mo−dithiolene complexes 21,24,26 in terms of the metrical parameters associated with the Tp* ligand and inner coordination sphere bond distances and angles. However, conformational differences between 2 and 4 are observed when comparing the dihedral angle between the quinoxaline and dithiolene planes. ...
Mononuclear Mo and W enzymes require a unique ligand known as molybdopterin (MPT). This ligand binds the metal through a dithiolene chelate, and the dithiolene bridges a reduced pyranopterin group. Pyran scission and formation have been proposed as a reaction of the MPT ligand that may occur within the enzymes to adjust reactivity at the Mo atom. We address this issue by investigating oxo-Mo(IV) model complexes containing dithiolenes substituted by pterin or quinoxaline and a hydroxyalkyl poised to form a pyran ring. While the pterin-dithiolene model complex exhibits a low energy, reversible pyran cyclization, here we report that pyran cyclization does not spontaneously occur in the quinoxalyl-dithiolene model. However, protonating the quinoxalyl-dithiolene model induces pyran cyclization forming an unstable, pyrano-quinoxalyl-dithiolene complex which subsequently dehydrates and rearranges to a pyrrolo-quinoxlyl-dithiolene complex that was previously characterized. The protonated pyrano-quinoxalyl-dithiolene complex was characterized by absorption spectroscopy and cyclic voltammetry, and these results suggest pyran cyclization leads to a significant change in the Mo electronic structure exhibited as a strong intraligand charge transfer (ILCT) transition and 370 mV positive shift of the Mo(V/IV) reduction potential. The influence of protonation on quinoxaline reactivity supports the hypothesis that the local protein environment in the second coordination sphere of molybdenum cofactor (Moco) could control pyran cyclization. The results also demonstrate that the remarkable chemical reactivity of the pterin-dithiolene ligand is subtly distinct and not reproduced by the simpler quinoxaline analog that is often used to replace pterin in synthetic Moco models.
... 2,21−25 This "ligand folding" effect becomes more prominent as the d-electron count decreases (d 2 = 9°, d 1 = 35°, d 0 = 46°) 20 and allows for the holes created by lost electrons to be delocalized in metal− dithiolene orbitals. 20,22,26 These studies have formed the basis for a hypothesis asserting that the pyranopterin dithiolene ligand may be an active participant in the catalytic cycles of Mo/W enzymes by modulating active site redox potentials through static or dynamic changes in metal−dithiolene covalency. 24,25,27,28 For example, in the proposed catalytic cycle of sulfite oxidase the Mo center traverses through three different formal oxidation states during two sequential oneelectron oxidations of the Mo(IV) species back to the active Mo(VI) form. ...
... As such, the MLCT state closely mimics the Mo(IV) → Mo(V) electron-transfer process that occurs in the oxidative, electrontransfer half-reactions of XO family enzymes to ultimately regenerate the catalytically competent oxidized Mo(VI) state ( Figure 1A). Importantly, the only appreciable PDT con- 31,32 Removal of an electron from the donor orbital is anticipated to result in molecular distortions within the S−Mo−S dithiolene fragment, leading to rR enhancement of low-frequency molybdenum dithiolene core vibrations 31,32 that may be kinematically coupled to other low-frequency Moco modes. In this way, rR spectroscopy of wt-XDH and its Q197A and Q102G variants provides a means of making detailed lowfrequency Moco vibrational assignments and sensitively probing specific Moco−protein interactions. ...
... These have been assigned as arising from symmetric S−Mo−S stretching and bending modes, which have been observed to be mixed because of their similar frequencies. 31,32 The situation is quite different for XDH and XO, 20 where six low-frequency vibrations are observed ( Figure 3). Here, we consider the experimental rR data for these XDH Mo(IV)−P CT complexes in the context of the small-molecule analogue rR data and DFT frequency calculations. ...
... The 326 cm −1 mode (band C) observed for wt-XDH Mo(IV)−4-TV was also probed in earlier work that compared wt-XOR and wt-XDH rR spectra with both 4-TV-and 2,4-TVbound product molecules. 20 This mode is assigned as primarily arising from the symmetric S dithiolene −Mo−S dithiolene core stretching vibration by analogy to small-molecule oxomolybdenum dithiolene analogue compounds, 31,32 and this is additionally supported by comparison with our DFT frequency calculations. We note that a small 2 cm −1 shift to lower frequency is observed for this mode in the Q197A variant. ...
The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen-bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco-protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules. The resulting enzyme-product complexes possess intense near-IR absorption, allowing high-quality rR spectra to be collected on wt-XDH and the Q102G and Q197A variants. Small negative frequency shifts relative to wt-XDH are observed for the low-frequency Moco vibrations. These results are interpreted in the context of weak hydrogen-bonding and/or electrostatic interactions between Q102 and the -NH2 terminus of the PDT, and between Q197 and the terminal oxo of the Mo≡O group. The Q102A, Q102G, Q197A, and Q197E variants do not appreciably affect the kinetic parameters kred and kred/KD, indicating that a primary role for these glutamine residues is to stabilize and coordinate Moco in the active site of XO family enzymes but to not directly affect the catalytic throughput. Raman frequency shifts between wt-XDH and its Q102G variant suggest that the changes in the electron density at the Mo ion that accompany Mo oxidation during electron-transfer regeneration of the catalytically competent active site are manifest in distortions at the distant PDT amino terminus. This implies a primary role for the PDT as a conduit for facilitating enzymatic electron-transfer reactivity in xanthine oxidase family enzymes.
... The energy of this orbital can be altered through small perturbations in the geometry around the molybdenum center (e.g., changing the torsion angle of the coordinated amino acid, changing the fold angle of dithiolene ligand) and solvation of the center preferentially stabilizes the higher charges. [150][151][152][153] Both factors, the electronic structure dictated by the geometric structure and charge stabilization through solvation, are important in the Note that values in NR exhibit a larger spread, those of XO and SO are tightly clustered, with the exception of four points, which are representing substrate or inhibitor bound form or eukNR. ...
The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types - periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed.
... The description of the pterin in both structures as dihydropterin is consistent with bond distances in the pyrazine ring, where C17−N7 bond distances are typical for a CN bond while C22−N10 distances are longer, as expected for saturated bonds that result from −OH addition. Dithiolene Mo−S, S−C, and 16,17 with a very slight asymmetry observed within the chelate ring. The complexes are chiral in regard to whether the pterin extends to the right or left of the dithiolene chelate. ...
The syntheses and X-ray structures of two molybdenum pyranopterin dithiolene complexes in biologically relevant Mo(+4) and Mo(+5) states are reported. Crystallography reveals these complexes possess a pyran ring formed through a spontaneous cyclization reaction of a side-chain hydroxyl group at a C=N bond of the pterin. NMR data on the Mo(+4) complex suggests a reversible pyran ring cyclization in solution. These results provide experimental evidence that the pyranopterin dithiolene ligand in molybdenum and tungsten enzymes could be involved in dynamic processes modulated by the protein.
... A covalency induced "electronic buffer effect" has been observed in oxomolybdenumdithiolene model compounds. 62,63 This has been postulated to take advantage of the redox non-innocent behavior of the coordinated dithiolene to modulate 64 the reduction potential of the Mo site during the course of catalysis via Mo-dithiolene forward and back donation. In the Mo hydroxylases, we find that similar charge transfer stabilizations occur along the reaction coordinate that effectively buffer the active site against the large formal charge changes that accompany C-H bond cleavage. ...
A detailed electron paramagnetic resonance (EPR) and computational study of a key paramagnetic form of xanthine oxidase (XO) has been performed and serves as a basis for developing a valence-bond description of C-H activation and transition-state (TS) stabilization along the reaction coordinate with aldehyde substrates. EPR spectra of aldehyde-inhibited XO have been analyzed in order to provide information regarding the relationship between the g, (95,97)Mo hyperfine (A(Mo)), and (13)C hyperfine (A(C)) tensors. Analysis of the EPR spectra has allowed for greater insight into the electronic origin of key delocalizations within the Mo-O(eq)-C fragment and how these contribute to C-H bond activation/cleavage and TS stabilization. A natural bond orbital analysis of the enzyme reaction coordinate with aldehyde substrates shows that both Mo═S π → C-H σ* (ΔE = 24.3 kcal mol(-1)) and C-H σ → Mo═S π* (ΔE = 20.0 kcal mol(-1)) back-donation are important in activating the substrate C-H bond for cleavage. Additional contributions to C-H activation derive from O(eq) lp → C-H σ* (lp = lone pair; ΔE = 8.2 kcal mol(-1)) and S lp → C-H σ* (ΔE = 13.2 kcal mol(-1)) stabilizing interactions. The O(eq)-donor ligand that derives from water is part of the Mo-O(eq)-C fragment probed in the EPR spectra of inhibited XO, and the observation of O(eq) lp → C-H σ* back-donation indicates a key role for O(eq) in activating the substrate C-H bond for cleavage. We also show that the O(eq) donor plays an even more important role in TS stabilization. We find that O(eq) → Mo + C charge transfer dominantly contributes to stabilization of the TS (ΔE = 89.5 kcal mol(-1)) and the Mo-O(eq)-C delocalization pathway reduces strong electronic repulsions that contribute to the classical TS energy barrier. The Mo-O(eq)-C delocalization at the TS allows for the TS to be described in valence-bond terms as a resonance hybrid of the reactant (R) and product (P) valence-bond wave functions.
