Biology subjects

Ragsdale, S. W.

Publications and source records attributed to Ragsdale, S. W..

8 recordsLinked to original sources

bioRxiv2026

Use of Substrate Analogues and X-ray spectroscopy Reveals an all Ferrous C-Cluster in CO Dehydrogenase

Carbon monoxide (CO) dehydrogenase (CODH) plays a key role in prokaryotic one-carbon metabolism by detoxifying CO and by driving CO2 reduction coupled to ATP production in the Wood-Ljungdahl Pathway. Here we focus on a Ni-Fe CODH (CODH-II), with an active site C-cluster, which is a [NiFe4S4] cluster arranged as a [NiFe3S4] subcluster with an additional, unique pendant Fe, e.g., [Fe3S4-Feu]. It catalyzes the reversible reduction of CO2 to CO without the requirement for an overpotential and with insignificant proton reduction. The redox states associated with catalysis are defined as Cred1 and Cred2. Despite crystal structures with near 1.0 A resolution, it has been a long-standing question where the electrons in these catalytically relevant redox states are stored within the C cluster. Using X-ray absorption spectroscopy (XAS), EPR, and substrate-analogue binding measurements, we clarify the electronic structure of these catalytically active states in addition to the resting state of CODH. We rule out recent postulates that catalysis involves a Ni0 state, a metal-metal bond, or a hydride intermediate. We reveal that CODH rests in the diamagnetic Cox form, which contains Ni2+ and an oxidized [Fe3S4-Feu]2+ cluster. Then, the C-cluster undergoes reductive activation on Fe to form paramagnetic Cred1, which binds CO and analog cyanide. Generation of Cred2, which binds CO2 and its analog cyanate, involves two sequential valence-localized electron transfers, generating Ni1+ and then [Fe3S4-Feu], forming an all-ferrous cluster. Our work sheds light on how CODH avoids the thermodynamically unfavorable generation of a CO2 radical anion intermediate formed in other catalytic systems by stabilizing electron density in the heterometallic C-cluster. We also highlight the importance of high-resolution XAS and use of substrate analogs to reveal the sequential, valence-localized electron transfers that occur during redox-dependent CODH catalysis. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/700957v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1e8d3caorg.highwire.dtl.DTLVardef@a89cc9org.highwire.dtl.DTLVardef@1cd569dorg.highwire.dtl.DTLVardef@1f1668c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Structure of active methyl-CoM reductase, Earth's main methane producer

Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the {beta}-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes.

biochemistry↗

bioRxiv2021

Heme oxygenase-2 (HO-2) binds and buffers labile heme, which is largely oxidized, in human embryonic kidney cells

Heme oxygenases (HO) detoxify heme by oxidatively degrading it into carbon monoxide, iron, and biliverdin, which is reduced to bilirubin and excreted. Humans express two isoforms: inducible HO-1, which is up-regulated in response to various stressors, including excess heme, and constitutive HO-2. While much is known about the regulation and physiological function of HO-1, comparatively little is known about the role of HO-2 in regulating heme homeostasis. The biochemical necessity for expressing constitutive HO-2 is largely dependent on whether heme is sufficiently abundant and accessible as a substrate under conditions in which HO-1 is not induced. By measuring labile heme, total heme, and bilirubin in human embryonic kidney HEK293 cells with silenced or over-expressed HO-2, and various HO-2 mutant alleles, we found that endogenous heme is too limiting to support HO-2 catalyzed heme degradation. Rather, we discovered that a novel role for HO-2 is to bind and buffer labile heme. Taken together, in the absence of excess heme, we propose that HO-2 regulates heme homeostasis by acting as a heme buffering factor in control of heme bioavailability. When heme is in excess, HO-1 is induced and both HO-2 and HO-1 can provide protection from heme toxicity by enzymatically degrading it. Our results explain why catalytically inactive mutants of HO-2 are cytoprotective against oxidative stress. Moreover, the change in bioavailable heme due to HO-2 overexpression, which selectively binds ferric over ferrous heme, is consistent with the labile heme pool being oxidized, thereby providing new insights into heme trafficking and signaling.

cell biology↗

bioRxiv2021

Nickel-Sulfonate Mode of Substrate Binding for Forward and Reverse Reactions of Methyl-SCoM Reductase Suggest a Radical Mechanism Involving Long Range Electron Transfer

Methyl-coenzyme M reductase (MCR) catalyzes both synthesis and anaerobic oxidation of methane (AOM). Its catalytic site contains Ni at the core of Cofactor F430. The Ni ion, in its low-valent Ni(I) state lights the fuse leading to homolysis of the C-S bond of methyl-coenzyme M (methyl-SCoM) to generate a methyl radical, which abstracts a hydrogen atom from Coenzyme B (HSCoB) to generate methane and the mixed disulfide CoMSSCoB. Direct reversal of this reaction activates methane to initiate anaerobic methane oxidation. Based on crystal structures, which reveal a Ni-thiol interaction between Ni(II)-MCR and inhibitor CoMSH, a Ni(I)-thioether complex with substrate methyl-SCoM has been transposed to canonical MCR mechanisms. Similarly, a Ni(I)-disulfide with CoMSSCoB is proposed for the reverse reaction. However, this Ni(I)-sulfur interaction poses a conundrum for the proposed hydrogen atom abstraction reaction because the >6 [A] distance between the thiol group of SCoB and the thiol of SCoM observed in the structures appears too long for such a reaction. Spectroscopic, kinetic, structural and computational studies described here establish that both methyl-SCoM and CoMSSCoB bind to the active Ni(I) state of MCR through their sulfonate groups, forming a hexacoordinate Ni(I)-N/O complex, not Ni(I)-S. These studies rule out direct Ni(I)-sulfur interactions in both substrate-bound states. As a solution to the mechanistic conundrum, we propose that both forward and reverse MCR reactions emanate through long-range electron transfer from Ni(I)-sulfonate complexes with methyl-SCoM or CoMSSCoB, respectively.

biochemistry↗

bioRxiv2020

13C Electron Nuclear Double Resonance Spectroscopy Shows Acetyl-CoA Synthase Binds Two Substrate CO in Multiple Binding Modes and Reveals the Importance of a CO-Binding 'Alcove'

EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and distal Nid. An alcove seen in the ACS crystal-structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO and is suggested to cradle this CO. Previously, we only knew WT ACS bound a single CO in the Ared-CO intermediate, here seen as forming Nip(I)-CO with CO on-axis of the dz2 odd-electron orbital (g{perp}>g||[~]2). The two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope now reveals a second CO bound in the dz2 orbitals equatorial plane. This WT A-cluster conformer dominates the nearly-conservative F229W variant, but 13C-ENDOR reveals a minority "A" conformation with (g||>g{perp}[~]2) characteristic of a cloverleaf (eg. dx2-y2) odd-electron orbital, and with Nip binding two, apparently in-plane CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni(I) [~]tenfold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the "A" conformation, but with [~]20% each of the WT conformer and an "O" state in which dz2 Nip(I) (g{perp}>g||[~]2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation.

biochemistry↗

bioRxiv2020

Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer

Rev-Erb{beta} is a nuclear receptor that couples circadian rhythm, metabolism, and inflammation.1-7 Heme binding to the protein modulates its function as a repressor, its stability, its ability to bind other proteins, and its activity in gas sensing.8-11 Rev-Erb{beta} binds Fe3+-heme tighter than Fe2+-heme, suggesting its activities may be regulated by the heme redox state.9 Yet, this critical role of heme redox chemistry in defining the proteins resting state and function is unknown. We demonstrate by electrochemical and whole-cell electron paramagnetic resonance experiments that Rev-Erb{beta} exists in the Fe3+ form within the cell essentially allowing the protein to be heme-replete even at low concentrations of labile heme in the nucleus. However, being in the Fe3+ redox state contradicts Rev-Erbs known function as a gas sensor, which dogma asserts must be a Fe2+ protein This paper explains why the resting Fe3+-state is congruent both with heme-binding and cellular gas sensing. We show that the binding of CO/NO elicits a striking increase in the redox potential of the Fe3+/Fe2+ couple, characteristic of an EC mechanism in which the unfavorable Electrochemical reduction of heme is coupled to the highly favorable Chemical reaction of gas binding, making the reduction spontaneous. Thus, Fe3+-Rev-Erb{beta} remains heme-loaded, crucial for its repressor activity, and only undergoes reduction when diatomic gases are present. This work has broad implications for hemoproteins where ligand-triggered redox changes cause conformational changes influencing proteins function or inter-protein interactions, like NCoR1 for Rev-Erb{beta}. This study opens up the possibility of CO/NO-mediated regulation of the circadian rhythm through redox changes in Rev-Erb{beta}.

biochemistry↗

bioRxiv2020

Absence of heme at the catalytic site of heme oxygenase-2 triggers its lysosomal degradation

Heme oxygenase-2 (HO2) and -1 (HO1) catalyze heme degradation to biliverdin, CO, and iron, forming an essential link in the heme metabolism network. Tight regulation of the cellular levels and catalytic activities of HO1 and HO2 is important for maintaining heme homeostasis. While transcriptional control of HO1 expression has been well-studied, how the cellular levels and activity of HO2 are regulated remains unclear. Here, the mechanism of post-translational regulation of cellular HO2 level by heme is elucidated. Under heme deficient conditions, HO2 is destabilized and targeted for degradation. In HO2, three heme binding sites are potential targets of heme-dependent regulation: one at its catalytic site; the others at its two heme regulatory motifs (HRMs). We report that, in contrast to other HRM-containing proteins, the cellular protein level and degradation rate of HO2 are independent of heme binding to the HRMs. Rather, under heme deficiency, loss of heme binding to the catalytic site destabilizes HO2. Consistently, a HO2 catalytic site variant that is unable to bind heme exhibits a constant low protein level and an enhanced protein degradation rate compared to the wild-type HO2. However, cellular heme overload does not affect HO2 stability. Finally, HO2 is degraded by the lysosome through chaperone-mediated autophagy, distinct from other HRM-containing proteins and HO1, which are degraded by the proteasome. These results reveal a novel aspect of HO2 regulation and deepen our understanding of HO2s role in maintaining heme homeostasis, paving the way for future investigation into HO2s pathophysiological role in heme deficiency response.

biochemistry↗

bioRxiv2019

Kinetics of enzymatic mercury methylation at nanomolar concentrations catalyzed by HgcAB

Methylmercury (MeHg) is a potent neurotoxin that bioaccumulates in fish. MeHg is generated by anaerobic bacteria and archaea possessing the gene pair hgcAB. Although bacterial mercury (Hg) methylation has been characterized in vivo, the specific role of HgcAB in catalyzing Hg methylation is not well understood. Here we report the kinetics of HgcAB-mediated Hg methylation in cell lysates of Desulfovibrio desulfuricans ND132 at nanomolar Hg concentrations. The enzymatic Hg methylation mediated by HgcAB is highly oxygen-sensitive, irreversible, and follows Michaelis-Menten kinetics with an apparent KM of 3.2 nM and Vmax of 19.7 fmol{middle dot}min-1{middle dot}mg-1 total protein for the substrate Hg(II). Although the abundance of HgcAB in the cell lysates is extremely low, Hg(II) was quantitatively converted to MeHg at subnanomolar substrate concentrations. Supplementation with ATP, methyltetrahydrofolate, or pyruvate did not enhance MeHg production under the experimental conditions. Insight into the kinetics of Hg methylation catalyzed by HgcAB advances our understanding of the complex global Hg cycle.

biochemistry↗