Structural insights into functional properties of the oxidized form of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes. The turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized to the metastable OH state, and a reductive phase, in which OH is reduced back to the R state. During each phase, two protons are translocated across the membrane. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX), we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide insights into the proton translocation mechanism of CcO.

Detection of a Geminate Photoproduct of Bovine Cytochrome c Oxidase by Time-Resolved Serial Femtosecond Crystallography.

Cytochrome c oxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, CcO has a unique binuclear center (BNC) composed of a copper atom (CuB) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCcO-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.

Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography.

Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu B ) and a heme a 3 iron, where O 2 binds and is reduced to water. CO is a versatile O 2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a 3 iron leads to a metastable intermediate (Cu B -CO), where CO is bound to Cu B , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a 3 iron and moved to a temporary binding site midway between the Cu B and the heme a 3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a 3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.

Structural basis for functional properties of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis1. The full turnover of the CcO reaction involves an oxidative phase, in which the reduced enzyme (R) is oxidized by molecular oxygen to the metastable oxidized OH state, and a reductive phase, in which OH is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes2. However, if OH is allowed to relax to the resting oxidized state (O), a redox equivalent to OH, its subsequent reduction to R is incapable of driving proton translocation2,3. How the O state structurally differs from OH remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX)4, we show that the heme a3 iron and CuB in the active site of the O state, like those in the OH state5,6, are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three CuB ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from OH, where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of CcO.

The Role of Heme Peroxo Oxidants in the Rational Mechanistic Modeling of Nitric Oxide Synthase: Characterization of Key Intermediates and Elucidation of the Mechanism.

Mammalian nitric oxide synthase (NOS) mediates the two-step O2 -dependent oxidative degradation of arginine, and has been linked to a medley of disease situations in humans. Nonetheless, its exact mechanism of action still remains unclear. This work presents the first NOS model system where biologically proposed heme superoxo and peroxo intermediates are assessed as active oxidants against oxime substrates. Markedly, heme peroxo intermediates engaged in a bioinspired oxime oxidation reaction pathway, converting oximes to ketones and nitroxyl anions (NO- ). Detailed thermodynamic, kinetic, and mechanistic interrogations all evince a rate-limiting step primarily driven by the nucleophilicity of the heme peroxo moiety. Coherent with other findings, 18 O and 15 N isotope substitution experiments herein suffice compelling evidence toward a detailed mechanism, which draw close parallels to one of the enzymatic proposals. Intriguingly, recent enzymatic studies also lend credence to these findings, and several relevant reaction intermediates have been observed during NOS turnover.

Temperature-dependent structural transition following X-ray-induced metal center reduction in oxidized cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in the inner membrane of mitochondria. It contains four metal redox centers, two of which, CuB and heme a3, form the binuclear center (BNC), where dioxygen is reduced to water. Crystal structures of CcO in various forms have been reported, from which ligand-binding states of the BNC and conformations of the protein matrix surrounding it have been deduced to elucidate the mechanism by which the oxygen reduction chemistry is coupled to proton translocation. However, metal centers in proteins can be susceptible to X-ray-induced radiation damage, raising questions about the reliability of conclusions drawn from these studies. Here, we used microspectroscopy-coupled X-ray crystallography to interrogate how the structural integrity of bovine CcO in the fully oxidized state (O) is modulated by synchrotron radiation. Spectroscopic data showed that, upon X-ray exposure, O was converted to a hybrid O∗ state where all the four metal centers were reduced, but the protein matrix was trapped in the genuine O conformation and the ligands in the BNC remained intact. Annealing the O∗ crystal above the glass transition temperature induced relaxation of the O∗ structure to a new R∗ structure, wherein the protein matrix converted to the fully reduced R conformation with the exception of helix X, which partly remained in the O conformation because of incomplete dissociation of the ligands from the BNC. We conclude from these data that reevaluation of reported CcO structures obtained with synchrotron light sources is merited.

Indoleamine 2,3-dioxygenase (IDO)-1 and IDO-2 activity and severe course of COVID-19.

COVID-19 is a pandemic with high morbidity and mortality. In an autopsy cohort of COVID-19 patients, we found extensive accumulation of the tryptophan degradation products 3-hydroxy-anthranilic acid and quinolinic acid in the lungs, heart, and brain. This was not related to the expression of the tryptophan-catabolizing indoleamine 2,3-dioxygenase (IDO)-1, but rather to that of its isoform IDO-2, which otherwise is expressed rarely. Bioavailability of tryptophan is an absolute requirement for proper cell functioning and synthesis of hormones, whereas its degradation products can cause cell death. Markers of apoptosis and severe cellular stress were associated with IDO-2 expression in large areas of lung and heart tissue, whereas affected areas in brain were more restricted. Analyses of tissue, cerebrospinal fluid, and sequential plasma samples indicate early initiation of the kynurenine/aryl-hydrocarbon receptor/IDO-2 axis as a positive feedback loop, potentially leading to severe COVID-19 pathology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Proton-coupled electron transfer reactivities of electronically divergent heme superoxide intermediates: a kinetic, thermodynamic, and theoretical study.

Heme superoxides are one of the most versatile metallo-intermediates in biology, and they mediate a vast variety of oxidation and oxygenation reactions involving O2(g). Overall proton-coupled electron transfer (PCET) processes they facilitate may proceed via several different mechanistic pathways, attributes of which are not yet fully understood. Herein we present a detailed investigation into concerted PCET events of a series of geometrically similar, but electronically disparate synthetic heme superoxide mimics, where unprecedented, PCET feasibility-determining electronic effects of the heme center have been identified. These electronic factors firmly modulate both thermodynamic and kinetic parameters that are central to PCET, as supported by our experimental and theoretical observations. Consistently, the most electron-deficient superoxide adduct shows the strongest driving force for PCET, whereas the most electron-rich system remains unreactive. The pivotal role of these findings in understanding significant heme systems in biology, as well as in alternative energy applications is also discussed.

Characterization of the structural determinants of the ubiquitin-dependent proteasomal degradation of human hepatic tryptophan 2,3-dioxygenase.

Human hepatic tryptophan 2,3-dioxygenase (hTDO) is a homotetrameric hemoprotein. It is one of the most rapidly degraded liver proteins with a half-life (t1/2) of ∼2.3 h, relative to an average t1/2 of ∼2-3 days for total liver protein. The molecular mechanism underlying the poor longevity of hTDO remains elusive. Previously, we showed that hTDO could be recognized and ubiquitinated by two E3 ubiquitin (Ub) ligases, gp78/AMFR and CHIP, and subsequently degraded via Ub-dependent proteasomal degradation pathway. Additionally, we identified 15 ubiquitination K-sites and demonstrated that Trp-binding to an exosite impeded its proteolytic degradation. Here, we further established autophagic-lysosomal degradation as an alternative back-up pathway for cellular hTDO degradation. In addition, with protein kinases A and C, we identified 13 phosphorylated Ser/Thr (pS/pT) sites. Mapping these pS/pT sites on the hTDO surface revealed their propinquity to acidic Asp/Glu (D/E) residues engendering negatively charged DEpSpT clusters vicinal to the ubiquitination K-sites over the entire protein surface. Through site-directed mutagenesis of positively charged patches of gp78, previously documented to interact with the DEpSpT clusters in other target proteins, we uncovered the likely role of the DEpSpT clusters in the molecular recognition of hTDO by gp78 and plausibly other E3 Ub-ligases. Furthermore, cycloheximide-chase analyses revealed the critical structural relevance of the disordered N- and C-termini not only in the Ub-ligase recognition, but also in the proteasome engagement. Together, the surface DEpSpT clusters and the N- and C-termini constitute an intrinsic bipartite degron for hTDO physiological turnover.

Conformational Plasticity in Human Heme-Based Dioxygenases.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and human tryptophan dioxygenase (hTDO) are two important heme proteins that degrade the essential amino acid, l-tryptophan (Trp), along the kynurenine pathway. The two enzymes share a similar active site structure and an analogous catalytic mechanism, but they exhibit a variety of distinct functional properties. Here we used carbon monoxide (CO) as a structural probe to interrogate how the functionalities of the two enzymes are encoded in their structures. With X-ray crystallography, we detected an unexpected photochemical intermediate trapped in a crystal of the hIDO1-CO-Trp complex, where CO is photolyzed from the heme iron by X-rays at cryogenic temperatures (100 K). The CO photolysis triggers a large-scale migration of the substrate Trp, as well as the photolyzed CO, from the active site to a temporary binding site, Sa*. It is accompanied by a large conformational change to an active site loop, JK-LoopC, despite the severely restricted protein motion under the frozen conditions, which highlights the remarkable conformational plasticity of the hIDO1 protein. Comparative studies of a crystal of the hTDO-CO-Trp complex show that CO and Trp remain bound in the active site under comparable X-ray illumination, indicating a much more rigid protein architecture. The data offer important new insights into the structure and function relationships of the heme-based dioxygenases and provide new guidelines for structure-based design of inhibitors targeting them.

Structural Basis of Inhibitor Selectivity in Human Indoleamine 2,3-Dioxygenase 1 and Tryptophan Dioxygenase.

Indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) are two of the only three heme-based dioxygenases in humans. They have recently been identified as key cancer immunotherapeutic drug targets. While structures of hIDO1 in complex with inhibitors have been documented, so far there are no structures of hTDO-inhibitor complexes available. Here we use PF-06840003 (IPD), a hIDO1-selective inhibitor in clinical trials, as a structural probe to elucidate inhibitor-selectivity in hIDO1 versus hTDO. Spectroscopic studies show that IPD exhibits 400-fold higher inhibition activity toward hIDO1 with respect to hTDO. Crystallographic structures reveal that the binding pocket of IPD in the active site in hIDO1 is much more flexible as compared to that in hTDO, which offers a molecular explanation for the superior inhibition activity of IPD in hIDO1 with respect to hTDO. In addition to the IPD bound in the active site, a second IPD molecule was identified in an inhibitory site on the proximal side of the heme in hIDO1 and in an exosite that is ∼40 Šaway from the active site in hTDO. Taken together the data provide new insights into structure-based design of mono and dual inhibitors targeting hIDO1 and/or hTDO.

Snapshot of an oxygen intermediate in the catalytic reaction of cytochrome c oxidase.

Cytochrome c oxidase (CcO) reduces dioxygen to water and harnesses the chemical energy to drive proton translocation across the inner mitochondrial membrane by an unresolved mechanism. By using time-resolved serial femtosecond crystallography, we identified a key oxygen intermediate of bovine CcO. It is assigned to the PR-intermediate, which is characterized by specific redox states of the metal centers and a distinct protein conformation. The heme a3 iron atom is in a ferryl (Fe4+ = O2-) configuration, and heme a and CuB are oxidized while CuA is reduced. A Helix-X segment is poised in an open conformational state; the heme a farnesyl sidechain is H-bonded to S382, and loop-I-II adopts a distinct structure. These data offer insights into the mechanism by which the oxygen chemistry is coupled to unidirectional proton translocation.

Diaryl hydroxylamines as pan or dual inhibitors of indoleamine 2,3-dioxygenase-1, indoleamine 2,3-dioxygenase-2 and tryptophan dioxygenase.

Tryptophan (Trp) catabolizing enzymes play an important and complex role in the development of cancer. Significant evidence implicates them in a range of inflammatory and immunosuppressive activities. Whereas inhibitors of indoleamine 2,3-dioxygenase-1 (IDO1) have been reported and analyzed in the clinic, fewer inhibitors have been described for tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase-2 (IDO2) which also have been implicated more recently in cancer, inflammation and immune control. Consequently the development of dual or pan inhibitors of these Trp catabolizing enzymes may represent a therapeutically important area of research. This is the first report to describe the development of dual and pan inhibitors of IDO1, TDO and IDO2.

Mapping the Binding Trajectory of a Suicide Inhibitor in Human Indoleamine 2,3-Dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an important heme-containing enzyme that is a key drug target for cancer immunotherapy. Several hIDO1 inhibitors have entered clinical trials, among which BMS-986205 (BMS) stands out as the only suicide inhibitor. Despite its "best-in-class" activity, the action mechanism of BMS remains elusive. Here, we report three crystal structures of hIDO1-BMS complexes that define the complete binding trajectory of the inhibitor. BMS first binds in a solvent exposed surface cleft near the active site in an extended conformation. The initial binding partially unfolds the active site, which triggers heme release, thereby exposing a new binding pocket. The inhibitor then undergoes a large scale movement to this new binding pocket, where it binds by adopting a high energy kinked conformation. Finally, the inhibitor relaxes to a bent conformation, via an additional large scale rearrangement, culminating in the energy minimum state. The structural data offer a molecular explanation for the remarkable efficacy and suicide inhibition activity of the inhibitor. They also suggest a novel strategy that can be applied for drug development targeting hIDO1 and related enzymes.

Inhibition Mechanisms of Human Indoleamine 2,3 Dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) and tryptophan dioxygenase (hTDO) catalyze the same dioxygenation reaction of Trp to generate N-formyl kynurenine (NFK). They share high structural similarity, especially in the active site. However, hIDO1 possesses a unique inhibitory substrate binding site (Si) that is absent in hTDO. In addition, in hIDO1, the indoleamine group of the substrate Trp is H-bonded to S167 through a bridging water, while that in hTDO is directly H-bonded to H76. Here we show that Trp binding to the Si site or the mutation of S167 to histidine in hIDO1 retards its turnover activity and that the inhibited activity can be rescued by an effector, 3-indole ethanol (IDE). Kinetic studies reveal that the inhibited activity introduced by Trp binding to the Si site is a result of retarded recombination of the ferryl moiety with Trp epoxide to form NFK and that IDE reverses the effect by preventing Trp from binding to the Si site. In contrast, the abolished activity induced by the S167H mutation is primarily a result of ∼5000-fold reduction in the O2 binding rate constant, possibly due to the blockage of a ligand delivery tunnel, and that IDE binding to the Si site reverses the effect by reopening the tunnel. The data offer new insights into structure-based design of hIDO1-selective inhibitors.

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1.

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near CuB, which, in turn, moves closer to the heme a3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3, thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.

Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth.

We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.

Reaction intermediates in the heme degradation reaction by HutZ from Vibrio cholerae.

HutZ is a heme-degrading enzyme in Vibrio cholerae. It converts heme to biliverdin via verdoheme, suggesting that it follows the same reaction mechanism as that of mammalian heme oxygenase. However, none of the key intermediates have been identified. In this study, we applied steady-state and time-resolved UV-vis absorption and resonance Raman spectroscopy to study the reaction of the heme-HutZ complex with H2O2 or ascorbic acid. We characterized three intermediates: oxyferrous heme, meso-hydroxyheme, and verdoheme complexes. Our data support the view that HutZ degrades heme in a manner similar to mammalian heme oxygenase, despite their low sequence and structural homology.

A Comparative Analysis of the Effector Role of Redox Partner Binding in Bacterial P450s.

The camphor monooxygenase, cytochrome P450cam, exhibits a strict requirement for its own redox partner, putidaredoxin (Pdx), a two-iron-sulfur ferredoxin. The closest homologue to P450cam, CYP101D1, is structurally very similar, uses a similar redox partner, and exhibits nearly identical enzymatic properties in the monooxygenation of camphor to give the same single 5-exo-hydroxy camphor product. However, CYP101D1 does not strictly require its own ferredoxin (Arx) for activity because Pdx can support CYP101D1 catalysis but Arx cannot support P450cam catalysis. We have further examined the differences between these two P450s by determining the effect of spin equilibrium, redox properties, and stability of oxygen complexes. We find that Arx shifts the spin state equilibrium toward high-spin, which is the opposite of the effect of Pdx on P450cam. In both P450s, redox partner binding destabilizes the oxy-P450 complex but this effect is much weaker with CYP101D1. In addition, resonance Raman data show that structural perturbations observed in P450cam upon addition of Pdx are absent in CYP101D1. These data indicate that Arx does not play the same effector role in catalysis as Pdx does with P450cam. The most relevant structural difference between these two P450s centers on a catalytically important Asp residue required for proton-coupled electron transfer. We postulate that with P450cam larger Pdx-assisted motions are required to free this Asp for catalysis while the smaller number of restrictions in CYP101D1 precludes the need for redox partner-assisted structural changes.