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Structure of the immediate vicinity of the bound dioxygen . The distal pocket Gly residues and the bound dioxygen are shown in a CPK model, and the heme is illustrated in a ball-and-stick diagram. Carbon, nitrogen, and oxygen atoms of the heme and Gly residues are shown in gray, blue, and red, respectively. The bound dioxygen molecule is shown in magenta. Top, view from the pyrrole A; bottom, view from the distal side of the heme.
Source publication
HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen asso...
Contexts in source publication
Context 1
... and Gly-139 are tightly packed around bound O 2 (Fig. 4). The C atom of Gly-139 is 3 Å from the center of the O-O bond. The amide nitrogen of Gly-139 is located within hydrogen bonding distance to the oxygen atom bound to the heme iron (3 Å). The carbonyl oxygen of Gly-135 is also very close to both oxygen atoms (3 Å). This tight packing by the two Gly residue points bound O 2 directly ...
Context 2
... crystal structure of the oxy form of the heme-HmuO com- plex confirms this view and shows that the two distal Gly resi- dues at the "kink" in the distal helix direct the geometry of the Fe-O-O complex for regiospecific hydroxylation of the -meso- carbon atom of the heme group (Fig. 4). A small cleft created by two distal Gly residues is present in the ligand-free ferrous com- plex of HmuO (14), and O 2 binds to the heme iron without significant rearrangement of this distal pocket structure (Fig. 3). The steric pressure imposed by these two Gly residues is respon- sible for the acute Fe-O 2 bending angle and the ...
Citations
... The known structures include human HO-1 and HO-2 [18][19][20][21][22][23], rat HO-1 [24][25][26][27], two cyanobacterial HOs from Synechocystis sp. PCC 6803, SynHO-1 and SynHO-2 [28,29], and four pathogenic bacterial HOs, HemO from Neisseria meningitidis [30], Pseudomonas aeruginosa HO [31], HmuO from Corynebacterium diphtheriae [32][33][34], and Leptospira interrogans HO (LiHO) [35]. Mammalian HOs use NADPHcytochrome P450 reductase (CPR) as redox partner to obtain electrons from NADPH, and the electron transfer path has been revealed by the complex structures of rat CPR-HO-1 [36,37]. ...
Arabidopsis thaliana heme oxygenase‐1 (AtHO‐1), a metabolic enzyme in the heme degradation pathway, serves as a prototype for study of the bilin‐related functions in plants. Past biological analyses revealed that AtHO‐1 requires ferredoxin‐NADP+ reductase (FNR) and ferredoxin for its enzymatic activity. Here, we characterized the binding and degradation of heme by AtHO‐1, and found that ferredoxin is a dispensable component of the reducing system that provides electrons for heme oxidation. Furthermore, we reported the crystal structure of heme‐bound AtHO‐1, which demonstrates both conserved and previously undescribed features of plant heme oxygenases. Finally, the electron transfer pathway from FNR to AtHO‐1 is suggested based on the known structural information.
... Biliverdin is subsequently converted to bilirubin by biliverdin reductase [6][7][8]. Almost all crystal structures of HO complexed with its substrates, reaction intermediates, and product have been determined [9][10][11][12][13][14][15][16][17][18][19]. ...
Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme–HO-1, whereas the reduction in heme–HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme–HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme–HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme–HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme–HO-1 in solution. We conclude that the “open–close” transition of CPR is indispensable for electron transfer from CPR to heme–HO-1.
... These hydrogen bonds increased O 2 affinity for the heme-HO complex. Similar binding of O 2 to the heme-HO complex derived from Corynebacterium diphtheriae, heme-HmuO, was observed in a crystallographic study [64]. Substrate O 2 was similarly bound to the heme iron; and the angle of Fe-O-O was 101-114°. ...
... The crystal structures of the apo form, O 2 -bound form, verdoheme-bound form, and biliverdin-bound form were also reported using HO from Corynebacterium diphtheria [60,64,96,97]. These structures are similar to those of mammalian HOs. ...
In mammals, catabolism of the heme group is indispensable for life. Heme is first cleaved by the enzyme heme oxygenase (HO) to the linear tetrapyrrole biliverdin IXα (BV), and BV is then converted to bilirubin by biliverdin reductase (BVR). HO utilizes three oxygen molecules (O2) and seven electrons supplied by NADPH-cytochrome P450 oxidoreductase (CPR) to open the heme ring and BVR reduces BV through the use of NAD(P)H. Structural studies of HOs, including substrate-bound, reaction intermediate-bound, and several specific inhibitor-bound forms, reveal details explaining of substrate binding to HO and mechanisms underlying-specific HO reaction progression. Cryo-trapped structures and a time-resolved spectroscopic study examining photolysis of the bond between the distal ligand and heme iron demonstrate how CO, produced during the HO reaction, dissociates from the reaction site with a corresponding conformational change in HO. The complex structure containing HO and CPR provides details of how electrons are transferred to the heme-HO complex. Although the tertiary structure of BVR and its complex with NAD+ was determined more than 10 years ago, the catalytic residues and the reaction mechanism of BVR remain unknown. A recent crystallographic study examining cyanobacterial BVR in complex with NADP+ and substrate BV provided some clarification regarding these issues. Two BV molecules are bound to BVR in a stacked manner, and one BV may assist in the reductive catalysis of the other BV. In this review, recent advances illustrated by biochemical, spectroscopic, and crystallographic studies detailing the chemistry underlying the molecular mechanism of HO and BVR reactions are presented.
... A relatively low ν(OeO) stretching mode seen near 1140 cm − 1 in the rR spectrum [282,283] is indicative of a ferric superoxide (Fe 3 + -O 2 − ) species. The FeeOeO fragment is inherently bent, with the FeeOeO angle being 130 o -140°, as observed by X-ray crystallography in CYP101A1 [284,285], similar to those observed in myoglobin (122 o ) [286], hemoglobin (124 o and 126 o in αand β-subunits) [287], cytochrome c peroxidase (133 o ) [288], horseradish peroxidase (126 o ) [289], guanylate cyclase H-NOX domain (114°-134°) [290], FixL heme domain (119°) [291] and heme oxygenase (110°) [292]. Subsequently, the Xray structures of oxy-ferrous complexes in mutant CYP101A1 [285], CYP107A1 [293] and CYP158A2 [294] were solved. ...
The cytochrome P450 monooxygenases (P450s) are thiolate heme proteins that can, often under physiological conditions, catalyze many distinct oxidative transformations on a wide variety of molecules, including relatively simple alkanes or fatty acids, as well as more complex compounds such as steroids and exogenous pollutants. They perform such impressive chemistry utilizing a sophisticated catalytic cycle that involves a series of consecutive chemical transformations of heme prosthetic group. Each of these steps provides a unique spectral signature that reflects changes in oxidation or spin states, deformation of the porphyrin ring or alteration of dioxygen moieties. For a long time, the focus of cytochrome P450 research was to understand the underlying reaction mechanism of each enzymatic step, with the biggest challenge being identification and characterization of the powerful oxidizing intermediates. Spectroscopic methods, such as electronic absorption (UV–Vis), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), electron nuclear double resonance (ENDOR), Mossbauer, X-ray absorption (XAS), and resonance Raman (rR), have been useful tools in providing multifaceted and detailed mechanistic insights into the biophysics and biochemistry of these fascinating enzymes. The combination of spectroscopic techniques with novel approaches, such as cryoreduction and Nanodisc technology, allowed for generation, trapping and characterizing long sought transient intermediates, a task that has been difficult to achieve using other methods. Results obtained from the UV–Vis, rR and EPR spectroscopies are the main focus of this review, while the remaining spectroscopic techniques are briefly summarized. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
... The mechanisms of proton delivery are poorly understood. They are likely to involve networks of hydrogen-bonded residues and water molecules that connect the distal iron ligands all the way to the solvent surface [28][29][30][31][32] . As further new information emerges, the data presented here will help to lay a foundation for understanding the subtle and varied mechanisms that control proton delivery and biological reactivity in cytochrome P450 and other catalytic heme enzymes. ...
Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)–OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)–OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)–OH in a peroxidase.
... 62 The structure of the bacterial Fe II −O 2 heme−HmuO complex confirmed that the 110°Fe− O−O bond angle is due to interaction of the O 2 ligand with the distal helix ( Figure 3B). 63 Reduction of the Fe II −O 2 complex to the activated Fe III −OOH complex leads to the formation of α- meso-hydroxyheme, 64 which in the presence of oxygen is rapidly converted to ferric verdoheme. 41 In contrast, the noncanonical enzymes degrade heme to bilin products distinct from those for the canonical enzymes in which either the meso-carbon is retained as an aldehyde (as in MhuD) or released as formaldehyde (as in IsdG/I). ...
... 74 Collectively, the data support a theory wherein the water promotes nucleophilic attack by acting as a hydrogen-bond acceptor 73 or alternatively constrains the bent end-on geometry of Fe III −OOH for interaction with the α-meso-carbon. 63,74 Furthermore, while the conserved Asp is absent in the N. meningitides and P. aeruginosa HemOs, the hydrogen-bonding network is conserved. 53,55 In contrast to the water-driven oxygen activation of the Fe III −OOH intermediate, Rivera and co-workers proposed that the initial hydroxylation was facilitated by the heme. ...
Conspectus The eukaryotic heme oxygenases (HOs) (E.C. 1.14.99.3) convert heme to biliverdin, iron, and carbon monoxide (CO) in three successive oxygenation steps. Pathogenic bacteria require iron for survival and infection. Extracellular heme uptake from the host plays a critical role in iron acquisition and virulence. In the past decade, several HOs required for the release of iron from extracellular heme have been identified in pathogenic bacteria, including Corynebacterium diphtheriae, Neisseriae meningitides, and Pseudomonas aeruginosa. The bacterial enzymes were shown to be structurally and mechanistically similar to those of the canonical eukaryotic HO enzymes. However, the recent discovery of the structurally and mechanistically distinct noncanonical heme oxygenases of Staphylococcus aureus and Mycobacterium tuberculosis has expanded the reaction manifold of heme degradation. The distinct ferredoxin-like structural fold and extreme heme ruffling are proposed to give rise to the alternate heme degradation products in the S. aureus and M. tuberculosis enzymes. In addition, several "heme-degrading factors" with no structural homology to either class of HOs have recently been reported. The identification of these "heme-degrading proteins" has largely been determined on the basis of in vitro heme degradation assays. Many of these proteins were reported to produce biliverdin, although no extensive characterization of the products was performed. Prior to the characterization of the canonical HO enzymes, the nonenzymatic degradation of heme and heme proteins in the presence of a reductant such as ascorbate or hydrazine, a reaction termed "coupled oxidation", served as a model for biological heme degradation. However, it was recognized that there were important mechanistic differences between the so-called coupled oxidation of heme proteins and enzymatic heme oxygenation. In the coupled oxidation reaction, the final product, verdoheme, can readily be converted to biliverdin under hydrolytic conditions. The differences between heme oxygenation by the canonical and noncanonical HOs and coupled oxidation will be discussed in the context of the stabilization of the reactive Fe(III)-OOH intermediate and regioselective heme hydroxylation. Thus, in the determination of heme oxygenase activity in vitro, it is important to ensure that the reaction proceeds through successive oxygenation steps. We further suggest that when bacterial heme degradation is being characterized, a systems biology approach combining genetics, mechanistic enzymology, and metabolite profiling should be undertaken.
... This indicates that HmuO most likely has a high affinity for oxygen, allowing it to scavenge trace amounts of oxygen to synthesize BV. In support of this conclusion, other work has shown that HmuO from Corynebacterium diphtheriae has a 20fold greater oxygen affinity than mammalian myoglobins, and heme oxygenases have been found in strict anaerobes, including Clostridium tetani and Clostridium perfringens, where they may function in maintaining an anoxic environment (30)(31)(32). HmuO was identified in the proteome of anaerobically grown R. palustris, which suggests that HmuO functions under culture conditions considered anaerobic (33). ...
Significance
Bacteriophytochromes (BphPs) are regulatory proteins that bind a light-absorbing chromophore called biliverdin. Recombinant BphPs show promise for use in regulating neuron function in mammals with light. We explored the possibility that BphPs may sense cues in addition to light. Our motivation was that biliverdin requires oxygen for its synthesis, and some bacteria use BphPs to control photosynthesis in the absence of oxygen. We found that the photosynthetic bacterium Rhodopseudomonas palustris requires two BphP proteins to sense low light when grown in the absence of oxygen; however, the BphPs do not need to have their chromophore to sense low light intensities. BphPs may respond to intracellular signals, such as reducing conditions, in addition to light to regulate downstream functions.
... A significant distinction between the canonical HO's and the P450/peroxidase monoxygenases is the absence of a residue providing the polar side chain that stabilizes the bound O 2 . The stabilization of the O 2 -ligand in HO is a combination of the distal helix conserved Gly-Gly motif and the extensive hydrogen bond network coordinated through bridging H 2 O molecules ( Fig. 3B) [28][29][30]38,39]. Furthermore, the absorption spectrum of the Fe(II)-O 2 heme HO-1 complex is similar to that of oxmyoglobin [33] and has a unique oxygen-isotope shift in the resonance Raman spectrum suggesting the bound O 2 is highly bent [40]. ...
... Furthermore, the absorption spectrum of the Fe(II)-O 2 heme HO-1 complex is similar to that of oxmyoglobin [33] and has a unique oxygen-isotope shift in the resonance Raman spectrum suggesting the bound O 2 is highly bent [40]. The structure of the Corynebacterium diphtheriae Fe(II)-O 2 heme-HmuO complex confirmed the highly bent OAO bond angle, a consequence of interactions with the distal helix Gly-Gly motif and the hydrogen bond network [39]. Indeed disruption of the hydrogen bond network in the mammalian HO-1 enzymes on mutation of Asp-140 resulted in destabilization of the Fe-OOH intermediate and accelerated decay to the Fe(IV)@O species [41,42]. ...
... Moreover, an electron density peak above the iron was tentatively interpreted to be a water molecule or hydroxide ion. Further insight into the nature of the Fe(II)-verdoheme binding site was revealed in the 1.7 Å structure of the Fe(II)-N 3 verdoheme HmuO complex indicating a highly bent Fe-N-N(-N) with an angle of $110°similar to that observed for the oxy-heme HmuO complex [39,81]. The distal N atom is directed toward the a-meso position through a hydrogen-bond interaction with the distal water, similar to that of the Fe(II)-O 2 heme complex. ...
... Since the first report of the crystal structure of human HO-1 (20), x-ray crystallographic analysis, together with spectroscopic and enzymatic studies, has made a considerable contribution in our elucidation of the HO catalytic mechanism (6,(21)(22)(23)(24)(25)(26). In HO catalysis, HO first binds heme to form a hemeenzyme complex, where the heme group is tightly sandwiched between the proximal and distal helices, the former of which includes the heme iron axial ligand His residue. ...
... Then O 2 binds to reduced penta-coordinate heme to form a meta-stable oxy complex. The crystal structure of the oxyheme-HmuO complex (26) reveals that the steric pressure of the distal helix realizes an acute Fe-O-O bond angle and directs the bound O 2 toward the porphyrin ␣-meso-carbon atom. The terminal oxygen atom forms a favorable hydrogen bond with a nearby water molecule that is a part of an extended solvent hydrogen bond network anchored by a conserved Asp residue in the distal heme pocket. ...
... One-electron reduction of Fe 3ϩ -biliverdin releases Fe 2ϩ , and the HO catalytic product, biliverdin, is generated (27). Although the structural insight into HO catalytic mechanism has been realized by the crystal structures (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)28), major questions remain to be answered in the substrate-free 4 and the product-bound forms of HO. ...
Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here we present crystal structures of the substrate-free, the Fe(3+)-biliverdin-bound, and the biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme-complex, move apart, and the proximal helix is partially unwounded. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the active site structure of the enzyme, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume usual helical conformation and are located in the heme pocket in the crystal structures of the Fe3+-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe(3+)-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe(3+)-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.
... (i) Steric constraints in the distal pocket steer the terminal OH moiety of the Fe 3+ -OOH oxidizing species toward the heme-meso carbon to be hydroxylated. Because the Fe 3+ -OOH intermediate is too reactive to be characterized structurally, evidence supporting this notion stems from the crystal structure of the Fe 2+ -O 2 complex in cd -HO [ 225 ] which shows that speci fi c interactions in the distal pocket direct the terminal O atom toward the heme a -meso carbon. Since the Fe 2+ -O 2 complex is the immediate precursor of the Fe 3+ -OOH oxidizing species, it is reasonable to assume that the terminal OH hydroxylates the a -meso carbon and therefore commits heme degradation toward the formation of a -biliverdin, the product of heme oxidation by cd -HO. ...
All but a few bacterial species have an absolute need for heme, and most are able to synthesize it via a pathway that is highly conserved among all life domains. Because heme is a rich source for iron, many pathogenic bacteria have also evolved processes for sequestering heme from their hosts. The heme biosynthesis pathways are well understood at the genetic and structural biology levels. In comparison, much less is known about the heme acquisition, trafficking, and degradation processes in bacteria. Gram-positive and Gram-negative bacteria have evolved similar strategies but different tactics for importing and degrading heme, likely as a consequence of their different cellular architectures. The differences are manifested in distinct structures for molecules that perform similar functions. Consequently, the aim of this chapter is to provide an overview of the structural biology of proteins and protein-protein interactions that enable Gram-positive and Gram-negative bacteria to sequester heme from the extracellular milieu, import it to the cytosol, and degrade it to mine iron.
