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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Conformational coupling between the active site and residues within the K(C)-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase.

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K(C)-channel is a conserved glutamate in subunit III. However, the majority of the K(C)-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K(C)-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b3. The data suggest a linkage between residues lining the K(C)-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241(Vc) and the active site cross-linked Y255(Vc), as well as two CuB histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.

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.

Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

Proton translocation in cytochrome c oxidase: insights from proton exchange kinetics and vibrational spectroscopy.

Cytochrome c oxidase is the terminal enzyme in the electron transfer chain. It reduces oxygen to water and harnesses the released energy to translocate protons across the inner mitochondrial membrane. The mechanism by which the oxygen chemistry is coupled to proton translocation is not yet resolved owing to the difficulty of monitoring dynamic proton transfer events. Here we summarize several postulated mechanisms for proton translocation, which have been supported by a variety of vibrational spectroscopic studies. We recently proposed a proton translocation model involving proton accessibility to the regions near the propionate groups of the heme a and heme a3 redox centers of the enzyme based by hydrogen/deuterium (H/D) exchange Raman scattering studies (Egawa et al., PLoS ONE 2013). To advance our understanding of this model and to refine the proton accessibility to the hemes, the H/D exchange dependence of the heme propionate group vibrational modes on temperature and pH was measured. The H/D exchange detected at the propionate groups of heme a3 takes place within a few seconds under all conditions. In contrast, that detected at the heme a propionates occurs in the oxidized but not the reduced enzyme and the H/D exchange is pH-dependent with a pKa of ~8.0 (faster at high pH). Analysis of the thermodynamic parameters revealed that, as the pH is varied, entropy/enthalpy compensation held the free energy of activation in a narrow range. The redox dependence of the possible proton pathways to the heme groups is discussed. This article is part of a Special Issue entitled: Vibrational spectroscopies and bioenergetic systems.

The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae.

The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQP(X)) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQP(X) and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the K(C)-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

Three-dimensional-printed gas dynamic virtual nozzles for x-ray laser sample delivery.

Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

Spectroscopic and mutagenesis studies of human PGRMC1.

Progesterone receptor membrane component 1 (PGRMC1) is a 25 kDa protein with an N-terminal transmembrane domain and a putative C-terminal cytochrome b5 domain. Heme-binding activity of PGRMC1 has been shown in various homologues of PGRMC1. Although the general definition of PGRMC1 is as a progesterone receptor, progesterone-binding activity has not been directly demonstrated in any of the purified PGRMC1 proteins fully loaded with heme. Here, we show that the human homologue of PGRMC1 (hPGRMC1) binds heme in a five-coordinate (5C) high-spin (HS) configuration, with an axial tyrosinate ligand, likely Y95. The negatively charged tyrosinate ligand leads to a relatively low redox potential of approximately -331 mV. The Y95C or Y95F mutation dramatically reduces the ability of the protein to bind heme, supporting the assignment of the axial heme ligand to Y95. On the other hand, the Y95H mutation retains ∼90% of the heme-binding activity. The heme in Y95H is also 5CHS, but it has a hydroxide axial ligand, conceivably stabilized by the engineered-in H95 via an H-bond; CO binding to the distal ligand-binding site leads to an exchange of the axial ligand to a histidine, possibly H95. We show that progesterone binds to hPGRMC1 and introduces spectral changes that manifest conformational changes to the heme. Our data offer the first direct evidence supporting progesterone-binding activity of PGRMC1.

Heme Stabilization of α-Synuclein Oligomers during Amyloid Fibril Formation.

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases.