Redox proteins

A central focus of protein science is in linking emerging knowledge of the physical, dynamic, and chemical features of proteins to biological pathways and disease states. Among biologically significant proteins are those engaged in the transfer of electrons, in some cases occurring as part of catalysis in enzymes, and in other instances involved in regulatory mechanisms orchestrating functional switching of proteins. This special issue on Redox Proteins brings together contributions from an array of disciplines, focusing on biochemical, structural, and computational approaches to reveal more about the way redox‐reactive proteins catalyze reactions and modulate functions in the broader biological context.

Redox reactions may be mediated by a bound cofactor, via amino acids comprising the protein itself, or by a synergy between the two. In terms of cofactors, several papers in this series describe diverse roles of flavins in enzymatic catalysis, where the protein provides crucial modulation of the reactivity of the bound isoalloxazine ring system. Inorganic‐centered redox cofactors are represented by contributions focused on the characterization of iron–sulfur clusters and on molybdopterins found in a number of molybdenum‐ and tungsten‐containing oxidoreductases. In other instances, the redox properties of a system derive from the protein itself; here several contributions describe the participation of the side chains of cysteine, methionine, or selenocysteine in catalysis and cellular signaling. The array of redox‐related topics forming this special issue reminds us of the fascinating breadth of chemistry and approaches that reflect modern research in redox biology.

Flavin‐linked enzymes from both pro‐ and eukaryotic sources and an array of reactions that they catalyze are the focus of a number of the contributions in this collection. Chenprakhon et al. (https://doi.org/10.1002/pro.3530) review the strategies for the flavin‐dependent monooxygenation of aromatic compounds. Within single‐component enzymes, the NADPH‐driven reduction of a tightly bound FAD prosthetic group is followed by the generation of a flavin C‐4a hydroperoxy adduct as a prelude to hydroxylation of the substrate. Two‐component systems comprise a reductase activity that catalyzes the NAD(P)H‐dependent reduction of flavins; the resulting dihydroflavins are then channeled, or freely diffuse, to the monooxygenase component for the aromatic oxygenation steps. In a bacterial two component system involved in the degradation of alkanesulfonates during sulfur deprivation, McFarlane et al. (https://doi.org/10.1002/pro.3504) investigate the role of a π‐helix in the NADPH:FMN reductase component. Robinson et al. (https://doi.org/10.1002/pro.3487) explore the mechanism of a fungal single‐component flavin‐dependent N‐monooxygenase that converts ornithine into the corresponding hydroxamate as the first committed step in the biosynthesis of a siderophore virulence factor. Another apparent monooxygenase, MICAL1 (molecule interacting with CasL), contains an FAD‐binding domain resembling p‐hydroxybenzoate hydroxylase. However, MICAL1 seems to function largely as an NADPH oxidase, generating hydrogen peroxide that serves as a modulator of intracellular function (Esposito et al. https://doi.org/10.1002/pro.3512). Here, the authors explore the binding of the small GTPase, Rab8, to MICAL1, and the consequent modulation of the flavin oxidase activity. Kean and Karplus (https://doi.org/10.1002/pro.3506) present the first crystal structure of a lactate monooxygenase. Their work describes a well‐ordered active site loop that confines the immediate products of flavin‐mediated oxidation, pyruvate and hydrogen peroxide, for subsequent reaction to yield acetate and carbon dioxide. In the disulfide‐generating flavoprotein oxidase QSOX1, Javitt et al. (https://doi.org/10.1002/pro.3537) explore the consequences of mutations that ablate a cis‐proline peptide bond in the vicinity of the redox‐active CxxC motif of the N‐terminal thioredoxin domain. The resulting gain‐of‐function mutation leads to a profound disruption of extracellular matrix function and to impaired fibroblast adhesion. Their work dovetails with a contribution by Fujimoto et al. (https://doi.org/10.1002/pro3530) that reviews a range of strategies to trap and identify protein substrates that interact with the redox‐active disulfide of thiol‐disulfide oxidoreductases. Hu et al. (https://doi.org/10.1002/pro.3479) describe an FMN‐dependent reductase that functions as an aerobic deiodinase. The varied modulation of flavin reactivity promoted by various halotyrosine derivatives is explored. Modulation of flavin reactivity is also a feature of a paper by Barber and Hondal (https://doi.org/10.1002/pro.3480). They found that the presence of selenocysteine in the C‐terminal shuttle disulfide of certain thioredoxin reductases leads to a marked enhancement of the ability of the bound FAD to promote 1‐electron chemistry. Ball et al. (https://doi.org/10.1002/pro.3514) have solved the crystal structure of a TIM‐barrel FMN‐dependent quinone oxidoreductase from Pseudomonas aeruginosa and show that the strict substrate specificity for NADH over NADPH reflects steric hindrance, rather than electrostatic repulsion. Koch et al. (https://doi.org/10.1002/pro.3517) describe an FAD‐linked lipoamide dehydrogenase‐like protein (IRC15; increased recombination centers 15) in Saccharomyces cerevisiae that lacks the redox‐active disulfide that normally communicates with bound flavin. They suggest that IRC15 serves as an NADH oxidase in vivo leading to hydrogen peroxide formation and the modulation of microtubule dynamics.

Metal‐containing redox centers, including iron, molybdenum, and tungsten, are also featured in four of the papers in this special issue. The critical 5′‐deoxyadenosyl radical participant in the radical S‐adenosylmethionine (SAM) superfamily is generated via reductive cleavage of SAM by at least one 4Fe–4S center. This reduction is ultimately driven by exogenous electron carriers, such as ferredoxins or flavodoxins. Two contributions show that all of the five ferredoxins from a hyperthermophile, Thermus maritima, can reduce MiaB, a radical SAM enzyme involved in methylthiolation of tRNA (Arcinas et al. https://doi.org/10.1002/pro.3548) and further characterize these ferredoxins electrochemically (Maiocco et al. https://doi.org/10.1002/pro.3547). In a third paper, Grell et al. (https://doi.org/10.1002/pro.3529) determine the crystal structure of a radical SAM enzyme, QueE, from Escherichia coli and discuss the factors modulating the reduction of its 4Fe‐4S center by flavodoxin. Finally, Niks and Hille (https://doi.org/10.1002/pro.3498) review structures and mechanisms of molybdenum‐ and tungsten‐containing enzymes that catalyze redox transformations between formate and carbon dioxide.

Cysteine serves as a versatile and under certain circumstances redox‐reactive catalytic residue in enzymes. In the case of peroxiredoxins, this residue is key to the reaction catalyzed, the reduction of hydroperoxide substrates (including peroxynitrite), but can also serve as a point of redox regulation. Two of the contributions to this issue address mechanistic aspects of peroxiredoxins. Dalla Rizza et al. (https://doi.org/10.1002/pro.3520) provide a comparison of various catalytic parameters of human Prdx1 and Prdx2, demonstrating essentially equal reactivity of the two toward hydrogen peroxide (rapidly producing sulfenic acid, R‐SOH, at the active site cysteine), yet considerably slower disulfide bond formation with the resolving cysteine for Prdx2 relative to Prdx1 (0.25 s⁻¹ vs. 11 s⁻¹ under the conditions used). This is consistent with the enhanced sensitivity of Prdx2 over Prdx1 toward hyperoxidation by peroxide substrates and its greater propensity (given the longer lifetime of the sulfenic acid) to form mixed disulfides with other proteins engaged in redox regulatory pathways. Feld et al. (https://doi.org/10.1002/pro.3490), studying the peroxiredoxin 6 of Plasmodium falciparum, report that the conserved histidyl residue proximal to the active site is catalytically non‐essential and that replacement by a tyrosine notably yields a more active enzyme.

In studying cysteine as a site of oxidative regulation in proteins, chemical and computational tools have been developed to evaluate the potential for specific sites to play a role in regulation. Computationally, disulfide bonds in structurally defined proteins can be analyzed for the strain they impart, and Haworth et al. (https://doi.org/10.1002/pro.3545) evaluate a type of “forbidden” disulfide that forms across strands of antiparallel β‐sheets, causing the β‐chains to tilt toward one another and imparting a significant strain on these proteins. Some of the chemical reagents that have found a use in detecting sites of cysteine oxidation in vitro or within cells are also a subject of contributions in this issue. The reagent dimedone, which has been known for decades to covalently modify cysteine sulfenic acids in proteins, was evaluated by Payne et al. (https://doi.org/10.1002/pro.3390) for its ability to react with and report on the oxidation at selenocysteine residues. They found that, while dimedone was able to label oxidized selenocysteine‐containing peptides at the putative selenenic acid moiety (R‐SeOH) in the absence of a nearby cysteine residue, the product was not stable and was readily reversed by thiols. Dimedone‐derived reagents were also used by Saez et al. (https://doi.org/10.1002/pro.3536) to demonstrate that sulforaphane, an anti‐inflammatory and anti‐cancer agent present in broccoli and other cruciferous vegetables, actually promotes chlamydial infections by suppressing protein oxidation in the mitochondria, presumably through its promotion of cellular antioxidant defenses.

Less information is available to date that firmly establishes methionine residues in proteins as bona fide sites of redox regulation, but in this issue, Tossounian et al. (https://doi.org/10.1002/pro.3440) provide evidence that elevated hydrogen peroxide levels elicit oxidation at the methionine sulfur of glutathione transferase Phi9 from Arabidopsis thaliana that decreases its transferase activity, particularly toward hydrophobic substrates engaged by the H‐site. This enzyme has no cysteine at all in its sequence.

In closing, this special issue highlights some of the diversity of current research on redox proteins – from fundamental aspects of enzymatic mechanism to the role of cysteine and selenium in cell signaling, and from methodologies to capture redox partners to a dissection of molybdopterin cofactor catalysis. We hope that these articles will stimulate new avenues of investigation in this vibrant and expanding field.