Proton transfer is essential in all multi-electron transformations. Identifying the proton donor in such processes is crucial to understanding the catalytic mechanism and optimizing activity through pKa matching, which can enhance proton transfer rates. However, aqueous solutions offer multiple proton sources, which confounds clear identification of the catalytically relevant proton donor. In this work, we utilize a model hydrogenase, nickel-substituted rubredoxin (NiRd), which is capable of electrocatalytic hydrogen evolution. Prior mechanistic studies suggested a proton transfer process was involved in the rate determining step on the basis of isotopic substitution and kinetic modeling. In this study, the identity of the proton donor in this rate-determining step was investigated by employing a range of buffers with varied pKa values. The impacts of concentration and pH on catalytic currents and turnover frequencies (TOF) were investigated. These results suggest that intermolecular, buffer-dependent proton transfer is not the rate-determining step for catalysis under typical electrocatalytic conditions. Quantitative electrochemical simulations incorporating buffer information allowed extraction of intrinsic thermodynamic and kinetic parameters for catalysis. Ultimately, mechanistic distinctions between the NiRd system and other hydrogen-evolving catalysts suggest that controlling the proton donor may serve as a handle for modulating activity in an intentional manner.
Certain Clostridium pasteurianum strains encode up to four FeFe-hydrogenases, which are homologous (identity > 20
Formate hydrogenlyases are metalloenzymes that can either produce molecular hydrogen gas or use H2 to convert carbon dioxide to formic acid, thus potentially contributing doubly to a sustainable energy future. The structure of formate hydrogenlyase reveals a membrane-bound redox enzyme that shares a common ancestor with the mitochondrial complex I (NADH dehydrogenase). As such, formate hydrogenlyase falls into a category of so‐called ‘complex‐I‐like’ enzymes, that are found in bacteria, archaea and eukaryotic organelles. They share a common core structure of a membrane arm (most likely involved in proton or ion translocation) and a peripheral arm containing metal cofactors and involved in electron transfer. In this work, we clone a gene cluster from Pectobacterium atrosepticum encoding formate hydrogenlyase-2 (FHL-2). A bank of Escherichia coli host strains, themselves devoid of various combinations of native formate hydrogenlyase genes, are employed to characterise FHL-2. We demonstrate that P. atrosepticum FHL-2 is active in an E. coli host in that it can generate H2 under fermentative growth conditions. Unlike native E. coli formate hydrogenlyase-1 (FHL-1), recombinant P. atrosepticum FHL-2 cannot perform the reverse reaction and generate formic acid from H2 and CO2. By testing different combinations of genes by taking an in vivo cross-complementation approach we conclude that the extended membrane arm exhibited by FHL-2 is a major factor in controlling directionality of the enzyme.
Electron-bifurcating flavoproteins split electron pairs and deposit them in spatially separated electron-acceptor pools. Just how electron-transfer networks regulate steady-state electron-bifurcation (EB) fluxes and efficiencies remains unclear. We model the steady-state kinetics of electron-bifurcation in NADH-dependent ferredoxin–NADP ^+ oxidoreductase I (Nfn1), based on the protein’s structure and electrochemistry. We use a many-particle master-equation to simulate the steady-state kinetics. The analysis identifies control points that are defined by electronic couplings between cofactors, and we explore how the interactions between cofactors influence EB function. We find that couplings on the low-potential branch dictate EB efficiency and the onset of short-circuiting, while the couplings on the high-potential branch determine the magnitude of the steady-state bifurcation flux in Nfn1. Analysis of interactions using the tunneling pathway model indicates that pathway connectivity between the bifurcating donor and its high-potential acceptor is weak for its distance; this decoupling may serve to further suppress short-circuiting and favor productive electron flow.
Protein and enzyme film electrochemistry is a powerful technique which enables the energetics and kinetics of biological electron transfer processes to be quantified. One of the major challenges is finding a suitable electrode surface upon which a redox active protein or enzyme can be adsorbed in an electroactive configuration. Multi-walled carbon nanotubes (MWCNTs) have been a useful class of electrode modifiers which facilitate electroactive protein adsorption and provide a substantial increase in electrode surface area. MWCNTs are therefore commonly used in bioelectrochemical studies, with applications in analytical electrochemistry and electrochemical sensing. Herein, we report the effect of electrochemical oxidation on a MWCNT-functionalised glassy carbon electrode in the presence of adsorbed protein. Previously uncharacterised redox functionalities are introduced by sweeping to oxidising potentials (> 0.8 V vs. SHE), with two reversible electron transfer signals subsequently appearing with midpoints of 80 and 390 mV at pH 7.0. This redox activity is hypothesised to be a result of the irreversible oxidation of the MWCNT-protein system resulting in the introduction of species capable of performing fast, reversible electron transfer.
Rhodoquinone (RQ) is a crucial electron carrier involved in anaerobic metabolism across select bacteria, protists, and animal species. Its biosynthesis is catalyzed by the rhodoquinone biosynthesis enzyme (RquA), a methyltransferase-like enzyme that uses S-adenosyl-L-methionine to transfer an amino group, converting ubiquinone (UQ) into RQ. The activity of RquA in vitro is enhanced by the presence of divalent metal cations. To probe the metal dependence of RquA, we characterized its interactions with Mn(II), Co(II), and Zn(II). We found that these metal cations bind to RquA with a 1:1 stoichiometry and that Mn(II) and Co(II) exhibited sub-micromolar binding affinities to RquA. Using Mn(II) as a spectroscopic probe, continuous‑wave electron paramagnetic resonance (EPR) revealed a single Mn(II) species with zero‑field splitting parameters |D| = 540(30) MHz and E/D = 0.30(3). Pulse EPR experiments on natural‑abundance and 15N‑labeled samples further identified a weakly‑to‑moderately coupled nitrogen ligand, with an isotropic hyperfine coupling constant |aiso(15N)| = 2.7(1) MHz. Integrating these data with an AlphaFold3‑derived structural model, we propose a putative binding site for the catalytically required divalent metal cation, providing new insight into the structural basis of RquA function.
Iron–ascorbate (Fe–Asc) is a clinically approved drug for iron-deficiency anemia (IDA), yet its formation and redox behavior under varying conditions remain poorly understood. Herein, we report the first comprehensive examination of Fe–Asc formation and redox reactivity in Tris buffer as a function of stoichiometry, pH, and oxygen availability. Oxidation of Fe(II) in air-saturated buffer accelerates with pH, yielding multiple species, primarily di- and tri-nuclear clusters. Notably, FeSO₄ with AscH produces a distinct purple complex (λmax = 510 nm), whose assembly depends critically on O₂, 1:3 stoichiometry of Fe: AscH, and buffer pH 7.5, whereas no such species forms anaerobically. ESI-MS suggests trinuclear clusters, whose stability and redox dynamics are monitored by time-resolved absorbance. The purple color disappears with dithionite or H₂O₂, but dithionite-treated samples regain color upon air exposure, demonstrating redox reversibility. Electrochemically, FeSO₄ shows cathodic (-0.32 V) and anodic (-0.06 V) peaks vs. Ag/AgCl, but the cathodic current decreases markedly in presence of AscH, reflecting Fe–Asc interactions. EPR spectra of FeSO₄ and FeSO₄/AscH are similar, with g = 9.43, 4.37 (high-spin Fe(III), S = 5/2) and g = 1.92 (S = ½, [Fe(III)–Fe(II)] coupling). These results suggest the formation of mixed valence trinuclear clusters with a dynamic ligand environment. The interaction between Fe and AscH with a specific stoichiometric ratio (Fe: AscH; 1:3), in Tris-buffer at pH7.5 under aerobic conditions, forms a purple complex, suggested as a mixed valence cluster, whereas anaerobic or dithionite-treated purple solutions yield a colorless solution that can reversibly interconvert with the purple form.
Iron–sulfur (Fe–S) clusters are common biological cofactors that facilitate vital redox reactions. Despite extensive research, the molecular basis of redox potential tuning in ferredoxin-like proteins remains an active area of debate. In this study, we combine statistical analysis of over one thousand [4Fe–4S]-containing protein structures from the Protein Data Bank (PDB) with broken-symmetry and extended broken-symmetry density functional theory to examine how cysteine ligand orientations and environmental screening affect redox properties of the clusters. We identified five main ligand configurations, three of which are predominant in natural structures. Among these, the adiabatic electron affinity differs by less than 0.1 V, indicating that, while geometry plays a secondary role, it allows localized fine-tuning of redox properties. In contrast, electrostatic and solvation effects primarily determine the overall potential range.
In this study, the thermodynamics of the one-electron (Fe2+/Fe3+) oxidation-reduction of the heme-bound fluoride complexes of adult hemoglobin (Hb-F) and horse heart myoglobin (Mb-F) were addressed to interpret the molecular changes these proteins undergo during the redox reactions. We measured the enthalpy (ΔHo’) and entropy (ΔSo’) of these reactions at pH 5 and pH 7, and also for the oxidation-reduction reactions of the metaquo complexes and for fluoride binding. The temperature dependence of the reduction potentials (Em) of Hb-F and Mb-F showed two redox pathways with distinct thermodynamic properties in the 5 to 45 °C temperature range, but only at pH 5. The Em-T plots of Hb-F and Mb-F were complementary to each other, with opposing signs in their ΔHo’ and ΔSo’. The redox reactions of the metaquo heme complexes and the heme-bound fluoride complexes (at pH 7) show no evidence of a bifurcated redox pathway. Given the similarities of the heme-ligand structures of oxy- and fluoride-bound complexes and comparable thermodynamics between oxidation and oxygen binding equilibrium, we theorize that one of the thermodynamic pathways is for stabilization of the heme-bound fluoride complex upon the redox change and the other is associated to a redox pathway that stabilizes the heme ferrous state, a route presumably associated to the unligated heme ferric-to-ferrous equilibrium. The serendipitous mixing of these redox pathways in the heme-bound fluoride complexes highlights the specificity of the heme proteins for oxygen binding and the possible use of these pathways for their respective physiological roles of storage and transport. The thermodynamics of the redox reactions of the heme-bound fluoride complexes of hemoglobin and myoglobin exhibit properties similar to those of their oxygen binding equilibria, which govern transport and oxygen storage. The thermodynamics of the redox reactions of the heme-bound fluoride complexes of hemoglobin and myoglobin exhibit properties similar to those of their oxygen binding equilibria, which govern transport and oxygen storage.
Here, we report a thioether-modified pyridine-2,6-dicarboxamide copper complex, [Cu(L)], 1 and its hydroxide-bridged dimer form, 2 exhibit different mechanisms of CO2 activation. The incorporation of a thioether moiety enhances the stabilization of the Cu(I) state and improves the redox reversibility of the complex for CO2 activation. While 1 monomer shows no direct reactivity toward CO2 in its resting state, the µ-OH dimer 2 reacts with CO2 to form a bicarbonate species, mimicking the nucleophilic CO2 hydration observed in Zn-containing carbonic anhydrases. In comparison, upon reduction, 1 forms a CO2-bound intermediate, revealing a redox-triggered pathway distinct from that of 2. Under electrochemical conditions, both complexes exhibit irreversible Cu(II/I) reduction in the presence of CO2, wherein an initial electron-transfer (E) step is followed by a chemical (C) transformation involving transient Cu-CO2 (EC-type) adduct formation. Overall, these findings demonstrate that the incorporation of soft sulfur donors equips copper complexes to mediate CO2 reactivity, providing a new direction toward the design of copper-based catalysts for CO2 conversion.
Hydrogen cyanide gas formation and subsequent contribution to inhalation injuries caused by residential and industrial fires is an increasing concern in public health care. Currently used cyanide antidotes are limited in their routes of administration and mechanisms of action. A potential cyanide antidote based on the [Mo2O2(μ-S)2]2+ core was assessed for its pharmacokinetic behavior and its efficacy in combating acute cyanide poisoning by inhalation in a mouse model. Pharmacokinetic studies revealed rapid absorption of the molybdenum compound into the bloodstream after intraperitoneal injection, and its complete elimination within eight hours. Prophylactic and therapeutic treatments with the compound by itself, and as a catalytic drug co-administered with thiosulfate, resulted in increased survival at lethal hydrocyanic acid concentrations.
Poly(ADP-ribose) polymerase 1 (PARP1) is a Zinc Finger (ZF) protein that is involved in DNA damage response. PARP1 contains three zinc finger (ZF) domains, two with a Cys2HisCys (CCHC) ligand set and one with a Cys4 (CCCC) ligand set that coordinate zinc. Persulfidation of cysteine residues in proteins is a new type of post-translational modification (PTM), for which ZFs are emerging as frequently modified. PARP1 has been identified as persulfidated in several cellular based persulfide-specific proteomics studies across several different cell types. Here, we examine PARP1 persulfidation by H2S. Two PARP1 peptide constructs, shPARP1-ZF1 and shPARP1-ZF2, were synthesized, purified, and shown to bind zinc and fold, using Co(II)/Zn(II) UV-visible (UV) monitored titrations and circular dichroism (CD) spectroscopy. The reactivity of the PARP ZFs with H2S was assessed using a UV-visible monitored 4-chloro-7-nitrobenzofurazan (NBF-Cl)/dimedone switch-tagging approach. Persulfidation for both PARP1 peptide constructs was observed under zinc-bound, oxygenated conditions. Superoxide intermediates were detected during the reaction using both dihydroethidine fluorescence and superoxide dismutase trapping. Together, these findings provide evidence that CCHC-type PARP ZFs can undergo persulfidation when Zn is bound and provide support for persulfidation being a general post-translational (PTM) modification for ZFs with different secondary structural elements.
In order to mitigate superoxide-induced damage, cells rely on superoxide dismutases (SODs), a family of metalloenzymes that catalyze the dismutation of superoxide. These enzymes display a positively-charged substrate entrance channel for superoxide guidance to the active metal center. ATCUN-like Ni(II) complexes have been shown to mimic NiSOD effectively and benefit from versatile peptide structures, which are ideal for introducing positive charges through arginine residues at various positions to modulate catalytic properties. We describe here five novel ATCUN-like Ni(II) complexes, with arginines at various positions in close proximity to the metal center. All the peptides form monometallic square planar Ni(II) complexes in water at pH 7.4, with either a N2S2 coordination (S-series) or an equilibrium between N2S2 and N3S1 coordination modes (N/S-series). The S-series demonstrates a clear beneficial effect of the charge increase from − 2 to + 2 on the pseudo-first order catalytic constant. Furthermore, the data obtained for the N/S complexes demonstrate that introducing an arginine residue in the central position of the ATCUN-like motif does not enhance activity, but actually impedes catalysis. These findings show that both the distance of the charged residues from the metal center and their positioning in space are of prime importance.
Iron-sulfur (Fe-S) clusters are ubiquitous as redox-active protein cofactors, but it is often difficult to collect protein structures in which redox centres are in uniform and well-defined oxidation states. Using spinach ferredoxin I (Fdx) as a model redox protein, we demonstrate an integrated methodological pathway for electrochemical modulation of redox state in protein crystals coupled with in crystallo EPR and online-UV-visible spectroscopy to verify oxidation state. We show that Fdx crystals can be electrochemically reduced, reversibly, without compromising lattice integrity or X-ray diffraction quality. We show that redox levels can be precisely ascertained in crystallo via EPR and UV-visible spectroscopy, enabling a direct correlation between protein structure and electronic state of the metal cluster. In this way, we generate and compare 'oxidised', 'reduced' and 're-oxidised' structures of Fdx. Overall, our approach demonstrates a pipeline which will be applicable to structure-function studies of a wide range of electron-transfer proteins and redox enzymes.
Cobalt is an essential trace element in biochemistry that plays a crucial role in the structure and function of several important biomolecules. In this review, vitamin B12 is discussed as one of the best-known examples in this area. Various forms of this vitamin, including methylcobalamin and adenosylcobalamin, play a crucial role in metabolic reactions in mammals and prokaryotes. It also discusses cobalt-containing enzymes that are essential for various biological processes. These enzymes are B12-dependent enzymes, which are well studied, and cobalt-containing enzymes, which are less well known, such as methionine aminopeptidase, nitrile hydratase, glucose isomerase, and prolidase. In addition to the significant role of cobalt complexes in biochemistry, these complexes are considered potent anticancer agents that can exert their antiproliferative effects through the production of ROS, cell cycle arrest, MMP breakdown, and induction of apoptosis in cancer cells. Cobalt complexes are also being explained here for their antimicrobial properties against a variety of pathogens, including bacteria, fungi, and viruses. Furthermore, examples of these complexes are presented as promising agents for the suppression of AD, which could be effective by binding to Aβ-peptides and preventing their aggregation, which is a central feature of the pathogenesis of AD, or by combating the oxidative damage associated with the disease, or even by interfering with the enzyme activities associated with this disease. Finally, the challenges related to the toxicity of cobalt and its compounds in medicine are discussed, and chelation therapy is considered an effective treatment for cobalt poisoning.
Cancer and tuberculosis remain among the leading causes of morbidity and mortality worldwide, and the emergence of drug resistance in both diseases highlights the urgent need for new therapeutic strategies. In this context, metal-based compounds combined with bioactive natural products have emerged as promising alternatives to conventional drugs. In the present study, a series of silver (I) complexes containing natural products (curcumin, lawsone, lapachol or alizarin) and triphenylphosphine (PPh3) (complexes 1–4) were synthesized and structurally characterized by IR spectroscopy, elemental analysis, molar conductivity, NMR (1H, 13C, 31P1H) spectroscopy and single crystal X-ray diffraction. The results of single crystal diffraction show that the two silver complexes are four-coordinated with two PPh3 and two O atoms of natural product, forming a distorted tetrahedral geometry. The in vitro cytotoxicity of silver(I) complexes was evaluated by MTT assay against four cancer cells and two normal cells, the result shows that the complexes presented a low IC50 value in general, including in normal cell lines. The complexes show better cytotoxic activity for A2780cis (cisplatin-resistant ovarian tumor cell line), being nearly 30 times higher than cisplatin. Silver complexes were tested against Mycobacterium tuberculosis (Mtb) reaching the threshold for effective antimicrobial activity. Furthermore, complex 4 showed good activity against clinical isolates of Mtb resistant to multiple first-line anti-TB drugs.
Xanthine oxidase plays a central role in purine catabolism in animals, plants and bacteria where it oxidises hypoxanthine to xanthine and xanthine to uric acid. In this work we have immobilised bovine xanthine oxidase on a glassy carbon electrode within a glutaraldehyde film and demonstrated hypoxanthine and xanthine oxidase electrocatalysis using mediator methylene blue as a mediator. The role of xanthine oxidase in the reduction of nitrite to nitric oxide has been probed in two ways. Using the electrochemically reduced mediator methyl viologen, nitrite reduction by xanthine oxidase is complicated by non-enzymatic reduction of nitrite by the mediator. Using hypoxanthine as reductant, nitrite reduction occurs but the product nitric oxide rapidly inactivates the enzyme. Xanthine oxidase can be restored to its functional form by redox cycling on an electrode in the presence of sulfide showing that nitric oxide leads to desulfuration of the molybdenum active site.
Arsenic is a dual-natured element, functioning as both a carcinogen and a therapeutic agent in cancer treatment. Arsenic trioxide is a clinically approved drug for acute promyelocytic leukaemia, administered either alone or in combination with all-trans retinoic acid. Additionally, arsenic trioxide and organic arsenic compounds, such as darinaparsin, show promising efficacy against various cancers. Given the multi-targeted nature of metallodrugs, a comprehensive understanding of their molecular mechanisms is critical for the rational design of more potent and less toxic therapeutics. In this review, we first outline the structures and activities of key arsenic-based anticancer agents, encompassing both inorganic and organic forms. We then provide a comprehensive overview of proteome-wide identification of arsenic-binding proteins in cancer cells, utilizing metalloproteomic approaches such as immobilized metal affinity chromatography, continuous-flow electrophoresis coupled with inductively coupled plasma mass spectrometry, biotinylated arsenical pull-down, and fluorescence-based metalloproteomics. Subsequently, we discuss how the identified arsenic proteome facilitates elucidation of the molecular mechanisms of arsenic trioxide and darinaparsin from a systems perspective. We also evaluate studies on the cytotoxicity of arsenic compounds at the single-cell level, highlighting their role in improving our understanding of the mode of action of a drug. Finally, we prospect future research direction in this field. This review aims to stimulate further investigation into the molecular mechanisms of arsenic-based drugs at a systems level, guiding the development of more effective and safer arsenic-based therapeutics for cancer treatment.
Hemoglobin and myoglobin are essential oxygen-binding proteins whose structural integrity is critical for proper function. This study performs atomic-level comparisons of wild-type and mutant forms to investigate how specific mutations impact oxygen-binding affinity. Substitution of the proximal histidine, responsible for coordinating the iron atom in the heme group, with tyrosine results shortening of bond distances in haemoglobin, while Van der Waals clashes in myoglobin; impairing function of both. Other mutations (α125Leu > Pro, β34Val > Asp, β63His > Tyr, β92His > Asn) compromise the protein stability by disrupting inter-chain hydrogen bonds and helical structures, reducing oxygen-binding efficiency. Cross-species comparisons, from Actinopterygii to Mammalia, underscore strong conservation of these critical residues essential for preserving the indispensable structural and functional integrity of these proteins since the dawn of their existence. Since the dawn of its existence high conservation of sequences especially histidine for maintaining the functionality of hemoglobin and myoglobin.
