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The principle of continuity posits that some central features of primordial biocatalytic mechanisms should still be present in the genetically dependent pathway of protein synthesis, a crucial step in the emergence of life. Key bimolecular reactions of this process are catalyzed by DNA-dependent RNA polymerases, aminoacyl-tRNA synthetases, and ribosomes. Remarkably, none of these biocatalysts contribute chemically active groups to their respective reactions. Instead, structural and functional studies have demonstrated that nucleotidic α-phosphate and β-d-ribosyl 2' OH and 3' OH groups can help their own catalysis, a process which, consequently, has been called "substrate-assisted". Furthermore, upon binding, the substrates significantly lower the entropy of activation, exclude water from these catalysts' active sites, and are readily positioned for a reaction. This binding mode has been described as an "entropy trap". The combination of this effect with substrate-assisted catalysis results in reactions that are stereochemically and mechanistically simpler than the ones found in most modern enzymes. This observation is consistent with the way in which primordial catalysts could have operated; it may also explain why, thanks to their complementary reactivities, β-d-ribose and phosphate were naturally selected to be the central components of early coding polymers.

RirA is a global iron regulator in diverse Alphaproteobacteria that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete conditions, RirA contains a [4Fe-4S] cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable [3Fe-4S] form that subsequently degrades to a [2Fe-2S] form and then to apo RirA, resulting in loss of high-affinity DNA-binding. The cluster is coordinated by three conserved cysteine residues and an unknown fourth ligand. Considering the lability of one of the irons and the resulting cluster fragility, we hypothesized that the fourth ligand may not be an amino acid residue. To investigate this, we considered that the introduction of an amino acid residue that could coordinate the cluster might stabilize it. A structural model of RirA, based on the Rrf2 family nitrosative stress response regulator NsrR, highlighted residue 8, an Asn in the RirA sequence, as being appropriately positioned to coordinate the cluster. Substitution of Asn8 with Asp, the equivalent, cluster-coordinating residue of NsrR, or with Cys, resulted in proteins that contained a [4Fe-4S] cluster, with N8D RirA exhibiting spectroscopic properties very similar to NsrR. The variant proteins retained the ability to bind RirA-regulated DNA, and could still act as repressors of RirA-regulated genes in vivo . However, they were significantly more stable than wild-type RirA when exposed to O 2 and/or low iron. Importantly, they exhibited reduced capacity to respond to cellular iron levels, even abolished in the case of the N8D version, and thus were no longer iron sensing. This work demonstrates the importance of cluster fragility for the iron-sensing function of RirA, and more broadly, how a single residue substitution can alter cluster coordination and functional properties in the Rrf2 superfamily of regulators., Competing Interests: The authors have no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Examination of the genetic code (GeCo) reveals that amino acids coded by (A/U) codons display a large functional spectrum and bind RNA whereas, except for Arg, those coded by (G/C) codons do not. From a stereochemical viewpoint, the clear preference for (A/U)-rich codons to be located at the GeCo half blocks suggests they were specifically determined. Conversely, the overall lower affinity of cognate amino acids for their (G/C)-rich anticodons points to their late arrival to the GeCo. It is proposed that i) initially the code was composed of the eight (A/U) codons; ii) these codons were duplicated when G/C nucleotides were added to their wobble positions, and three new codons with G/C in their first position were incorporated; and iii) a combination of A/U and G/C nucleotides progressively generated the remaining codons., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Several transcription factors of the Rrf2 family use an iron-sulfur cluster to regulate DNA binding through effectors such as nitric oxide (NO), cellular redox status and iron levels. [4Fe-4S]-NsrR from Streptomyces coelicolor (ScNsrR) modulates expression of three different genes via reaction and complex formation with variable amounts of NO, which results in detoxification of this gas. Here, we report the crystal structure of ScNsrR complexed with an hmpA1 gene operator fragment and compare it with those previously reported for [2Fe-2S]-RsrR/rsrR and apo-IscR/hyA complexes. Important structural differences reside in the variation of the DNA minor and major groove widths. In addition, different DNA curvatures and different interactions with the protein sensors are observed. We also report studies of NsrR binding to four hmpA1 variants, which indicate that flexibility in the central region is not a key binding determinant. Our study explores the promotor binding specificities of three closely related transcriptional regulators., (© 2022. The Author(s).)

The activation energy barrier of biochemical reactions is normally lowered by an enzyme catalyst, which directly helps the weakening of the bond(s) to be broken. In many metalloenzymes, this is a first coordination sphere effect. Besides having a direct catalytic action, enzymes can fix their reactive groups and substrates so that they are optimally positioned and also modify the water activity in the system. They can either activate substrates prior to their reaction or bind preactivated substrates, thereby drastically reducing local entropic effects. The latter type is well represented by some bisubstrate reactions, where they have been defined as "entropic traps". These can be described as " second coordination sphere " processes, but enzymes can also control the reactivity beyond this point through local conformational changes belonging to an " outer coordinate sphere " that can be modulated by substrate binding. We have chosen the [4Fe-4S] cluster-dependent enzyme quinolinate synthase to illustrate each one of these processes. In addition, this very old metalloenzyme shows low in vitro substrate binding specificity, atypical reactivity that produces dead-end products, and a unique modulation of its active site volume.

ATP is generally defined as the "energy currency" of the cell. Its phosphoanhydride P-O bonds are often considered to be "high energy" linkages that release free energy when broken, and its hydrolysis is described as "strongly exergonic". However, breaking bonds cannot release energy and ATP hydrolysis in motor and active transport proteins is not "strongly exergonic". So, the relevance of ATP resides elsewhere. As important as the nucleotide are the proteins that undergo functionally relevant conformational changes upon both ATP binding and release of ADP and inorganic phosphate. ATP phosphorylates proteins for signaling, active transport, and substrates in condensation reactions. The ensuing dephosphorylation has different consequences in each case. In signaling and active transport the phosphate group is hydrolyzed whereas in condensation reactions the phosphoryl fragment acts as a dehydrating agent. As it will be discussed in this article, ATP does much more than simply contribute free energy to biological processes., (© 2022 Wiley-VCH GmbH.)

Although nickel (Ni) is a minor element of the Earth's crust, it has played a major role in the evolution of life. This metal is a component of the active sites of several archaeal and bacterial anaerobic enzymes essential for bioenergy processes such as H2 and CO oxidation and CO2 fixation. Furthermore, Ni of meteoritic origin was probably involved in primordial organic phosphorylations. However, depending on its concentration, Ni can also be extremely toxic to most species. Through Earth's history, this paradoxical situation has provoked complex interactions between microorganisms, such as sulfate-reducing bacteria and the highly Ni-dependent methanogens. Ni-rich volcanic emissions have resulted in alterations of the biological carbon cycle caused by high archaeal production of greenhouse CH4 gas and the ensuing global temperature elevation. These emissions are also thought to have directly helped producing the most serious of the five major extinctions at the end of the Permian period., (© The Author(s) 2022. Published by Oxford University Press.)

Metalloproteins are involved in key cell processes such as photosynthesis, respiration, and oxygen transport. However, the presence of transition metals (notably iron as a component of [Fe-S] clusters) often makes these proteins sensitive to oxygen-induced degradation. Consequently, their study usually requires strict anaerobic conditions. Although X-ray crystallography has been the method of choice for solving macromolecular structures for many years, recently electron microscopy has also become an increasingly powerful structure-solving technique. We have used our previous experience with cryo-crystallography to develop a method to prepare cryo-EM grids in an anaerobic chamber and have applied it to solve the structures of apoferritin and the 3 [Fe 4 S 4 ]-containing pyruvate ferredoxin oxidoreductase (PFOR) at 2.40 Å and 2.90 Å resolution, respectively. The maps are of similar quality to the ones obtained under air, thereby validating our method as an improvement in the structural investigation of oxygen-sensitive metalloproteins by cryo-EM.

Quinolinate synthase, also called NadA, is a [4Fe-4S]-containing enzyme that uses what is probably the oldest pathway to generate quinolinic acid (QA), the universal precursor of the biologically essential cofactor nicotinamide adenine dinucleotide (NAD). Its synthesis comprises the condensation of dihydroxyacetone phosphate (DHAP) and iminoaspartate (IA), which involves dephosphorylation, isomerization, cyclization, and two dehydration steps. The convergence of the three homologous domains of NadA defines a narrow active site that contains a catalytically essential [4Fe-4S] cluster. A tunnel, which can be opened or closed depending on the nature (or absence) of the bound ligand, connects this cofactor to the protein surface. One outstanding riddle has been the observation that the so far characterized active site is too small to bind IA and DHAP simultaneously. Here, we have used site-directed mutagenesis, X-ray crystallography, functional analyses, and molecular dynamics simulations to propose a condensation mechanism that involves the transient formation of a second active site cavity to which one of the substrates can migrate before this reaction takes place.

Life requires energy to exist, to reproduce and to survive. Two major hypotheses have been put forward concerning the source of this energy at the very early stages of life evolution: (i) abiotic organics either brought to Earth by comets and/or meteorites, or produced at its atmosphere, and (ii) mineral surface-dependent bioinorganic catalytic reactions. Considering the latter possibility, I propose that, besides being a precursor of nucleic acids, adenosine triphosphate (ATP), which probably was used very early to improve the fidelity of nucleic acid polymerization, played an essential role in the transition between mineral-bound protocells and their free counterparts. Indeed, phosphorylation by ATP renders carboxylate groups electrophilic enough to react with nucleophiles such as amines, an effect that, thanks to their Lewis acid character, also have dehydrated metal ions on mineral surfaces. Early ATP synthesis for metabolic processes most likely depended on substrate level phosphorylation. However, the exaptation of a hexameric helicase-like ATPase and a transmembrane H + pump (which evolved to counteract the acidity caused by fermentation reactions within the protocell) generated a much more efficient membrane-bound ATP synthase that uses chemiosmosis to make ATP., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The [Fe 2 S 2 ]-RsrR gene transcription regulator senses the redox status in bacteria by modulating DNA binding, while its cluster cycles between +1 and +2 states-only the latter binds DNA. We have previously shown that RsrR can undergo remarkable conformational changes involving a 100° rotation of tryptophan 9 between exposed ( Out ) and buried ( In ) states. Here, we have used the chemical modification of Trp9, site-directed mutagenesis, and crystallographic and computational chemical studies to show that (i) the Out and In states correspond to oxidized and reduced RsrR, respectively, (ii) His33 is protonated in the In state due to a change in its p K a caused by cluster reduction, and (iii) Trp9 rotation is conditioned by the response of its dipole moment to environmental electrostatic changes. Our findings illustrate a novel function of protonation resulting from electron transfer.

Quinolinate synthase (NadA) is a [4Fe-4S] cluster-containing enzyme involved in the formation of quinolinic acid, the precursor of the essential NAD coenzyme. Here, we report the synthesis and activity of derivatives of the first inhibitor of NadA. Using multidisciplinary approaches we have investigated their action mechanism and discovered additional specific inhibitors of this enzyme.

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster between +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other. To our knowledge, this is the first example of a protein bound [Fe-S] cluster with three different amino acid side chains as ligands, and of Glu acting as ligand to a [2Fe-2S] cluster. Analyses of RsrR structures revealed a conformational change, centered on Trp9, which results in a significant shift in the DNA-binding helix-turn-helix region.

The origin of life is mostly divided into "genetics first" and "metabolism first" hypotheses. The former is based on spark-tube tests and organic species from meteorites and comets, and proposes a heterotrophic origin of life also consistent with the "RNA World" concept. The "metabolism first" hypothesis posits that life began autotrophically on minerals and/or hydrothermal vents. The lack of direct evidence means it is not possible to lend solid support to either hypothesis but the "metabolism first" option can be explored if a continuous geochemical, catalytically dynamic process is assumed. Using this approach, it is speculated that purine and pyrimidine synthesis originated on a mineral surface, which was later replaced by ATP. The same applies to redox processes where metal-bound hydrides could have been replaced by NAD., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The X-ray structure of an aerated achiral Ru-papain conjugate has revealed the hydroxylation of two tyrosine residues found near the ruthenium ion. The most likely mechanism involves a ruthenium-bound superoxide as the reactive species responsible for the first hydroxylation and the resulting high valent Ru(iv)[double bond, length as m-dash]O species for the second one.

Hydrogenases (H 2 ases) are benchmark electrocatalysts for H 2 production, both in biology and (photo)catalysis in vitro. We report the tailoring of a p-type Si photocathode for optimal loading and wiring of H 2 ase through the introduction of a hierarchical inverse opal (IO) TiO 2 interlayer. This proton-reducing Si|IO-TiO 2 |H 2 ase photocathode is capable of driving overall water splitting in combination with a photoanode. We demonstrate unassisted (bias-free) water splitting by wiring Si|IO-TiO 2 |H 2 ase to a modified BiVO 4 photoanode in a photoelectrochemical (PEC) cell during several hours of irradiation. Connecting the Si|IO-TiO 2 |H 2 ase to a photosystem II (PSII) photoanode provides proof of concept for an engineered Z-scheme that replaces the non-complementary, natural light absorber photosystem I with a complementary abiotic silicon photocathode., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The crystal structure of the Escherichia coli O2-sensitive C19G [NiFe]-hydrogenase-1 variant shows that the mutation results in a novel FeS cluster, proximal to the Ni-Fe active site. While the proximal cluster of the native O2-tolerant enzyme can transfer two electrons to that site, EPR spectroscopy shows that the modified cluster can transfer only one electron, this shortfall coinciding with O2 sensitivity. Computational studies on electron transfer help to explain how the structural and redox properties of the novel FeS cluster modulate the observed phenotype.

NadA is a multifunctional enzyme that condenses dihydroxyacetone phosphate (DHAP) with iminoaspartate (IA) to generate quinolinic acid (QA), the universal precursor of the nicotinamide adenine dinucleotide (NAD(P)) cofactor. Using X-ray crystallography, we have (i) characterized two of the reaction intermediates of QA synthesis using a "pH-shift" approach and a slowly reacting Thermotoga maritima NadA variant and (ii) observed the QA product, resulting from the degradation of an intermediate analogue, bound close to the entrance of a long tunnel leading to the solvent medium. We have also used molecular docking to propose a condensation mechanism between DHAP and IA based on two previously published Pyrococcus horikoshi NadA structures. The combination of reported data and our new results provide a structure-based complete catalytic sequence of QA synthesis by NadA.

Transmembrane NADPH oxidase (NOX) enzymes have been so far only characterized in eukaryotes. In most of these organisms, they reduce molecular oxygen to superoxide and, depending on the presence of additional domains, are called NOX or dual oxidases (DUOX). Reactive oxygen species (ROS), including superoxide, have been traditionally considered accidental toxic by-products of aerobic metabolism. However, during the last decade it has become evident that both O 2 •- and H 2 O 2 are key players in complex signaling networks and defense. A well-studied example is the production of O 2 •- during the bactericidal respiratory burst of phagocytes; this production is catalyzed by NOX2. Here, we devised and applied a novel algorithm to search for additional NOX genes in genomic databases. This procedure allowed us to discover approximately 23% new sequences from bacteria (in relation to the number of NOX-related sequences identified by the authors) that we have added to the existing eukaryotic NOX family and have used to build an expanded phylogenetic tree. We cloned and overexpressed the identified nox gene from Streptococcus pneumoniae and confirmed that it codes for an NADPH oxidase. The membrane of the S. pneumoniae NOX protein (SpNOX) shares many properties with its eukaryotic counterparts, such as affinity for NADPH and flavin adenine dinucleotide, superoxide dismutase and diphenylene iodonium inhibition, cyanide resistance, oxygen consumption, and superoxide production. Traditionally, NOX enzymes in eukaryotes are related to functions linked to multicellularity. Thus, the discovery of a large family of NOX-related enzymes in the bacterial world brings up fascinating questions regarding their role in this new biological context. IMPORTANCE NADPH oxidase (NOX) enzymes have not yet been reported in bacteria. Here, we carried out computational and experimental studies to provide the first characterization of a prokaryotic NOX. Out of 996 prokaryotic proteins showing NOX signatures, we initially selected, cloned, and overexpressed four of them. Subsequently, and based on preliminary testing, we concentrated our efforts on Streptococcus SpNOX, which shares many biochemical characteristics with NOX2, the referent model of NOX enzymes. Our work makes possible, for the first time, the study of pure forms of this important family of enzymes, allowing for biophysical and molecular characterization in an unprecedented way. Similar advances regarding other membrane protein families have led to new structures, further mechanistic studies, and the improvement of inhibitors. In addition, biological functions of these newly described bacterial enzymes will be certainly discovered in the near future., (Copyright © 2017 Hajjar et al.)

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe-4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe-4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove.

The enzyme NadA catalyzes the synthesis of quinolinic acid (QA), the precursor of the universal nicotinamide adenine dinucleotide (NAD) cofactor. Here, we report the crystal structures of complexes between the Thermotoga maritima (Tm) NadA K219R/Y107F variant and (i) the first intermediate (W) resulting from the condensation of dihydroxyacetone phosphate (DHAP) with iminoaspartate and (ii) the DHAP analogue and triose-phosphate isomerase inhibitor phosphoglycolohydroxamate (PGH). In addition, using the TmNadA K219R/Y21F variant, we have reacted substrates and obtained a crystalline complex between this protein and the QA product. We also show that citrate can bind to both TmNadA K219R and its Y21F variant. The W structure indicates that condensation causes dephosphorylation. We propose that catalysis by the K219R/Y107F variant is arrested at the W intermediate because the mutated protein is unable to catalyze its aldo-keto isomerization and/or cyclization that ultimately lead to QA formation. Intriguingly, PGH binds to NadA with its phosphate group at the site where the carboxylate groups of W also bind. Our results shed significant light on the mechanism of the reaction catalyzed by NadA.

The decahaem cytochrome MtrC from Shewanella oneidensis MR-1 was employed as a protein electron conduit between a porous indium tin oxide electrode and redox enzymes. Using a hydrogenase and a fumarate reductase, MtrC was shown as a suitable and efficient diode to shuttle electrons to and from the electrode with the MtrC redox activity regulating the direction of the enzymatic reactions.

The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electron-transfer processes at highly active and well-defined catalytic sites on a light-harvesting electrode surface. Herein, we report the integration of a hydrogenase on a TiO 2 -coated p-Si photocathode for the photo-reduction of protons to H 2 . The immobilized hydrogenase exhibits activity on Si attributable to a bifunctional TiO 2 layer, which protects the Si electrode from oxidation and acts as a biocompatible support layer for the productive adsorption of the enzyme. The p-Si|TiO 2 |hydrogenase photocathode displays visible-light driven production of H 2 at an energy-storing, positive electrochemical potential and an essentially quantitative faradaic efficiency. We have thus established a widely applicable platform to wire redox enzymes in an active configuration on a p-type semiconductor photocathode through the engineering of the enzyme-materials interface.

The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electron-transfer processes at highly active and well-defined catalytic sites on a light-harvesting electrode surface. Herein, we report the integration of a hydrogenase on a TiO2 -coated p-Si photocathode for the photo-reduction of protons to H2 . The immobilized hydrogenase exhibits activity on Si attributable to a bifunctional TiO2 layer, which protects the Si electrode from oxidation and acts as a biocompatible support layer for the productive adsorption of the enzyme. The p-Si|TiO2 |hydrogenase photocathode displays visible-light driven production of H2 at an energy-storing, positive electrochemical potential and an essentially quantitative faradaic efficiency. We have thus established a widely applicable platform to wire redox enzymes in an active configuration on a p-type semiconductor photocathode through the engineering of the enzyme-materials interface., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Carbon-sulfur bond formation at aliphatic positions is a challenging reaction that is performed efficiently by radical S-adenosyl-L-methionine (SAM) enzymes. Here we report that 1,3-thiazolidines can act as ligands and substrates for the radical SAM enzyme HydE, which is involved in the assembly of the active site of [FeFe]-hydrogenase. Using X-ray crystallography, in vitro assays and NMR spectroscopy we identified a radical-based reaction mechanism that is best described as the formation of a C-centred radical that concomitantly attacks the sulfur atom of a thioether. To the best of our knowledge, this is the first example of a radical SAM enzyme that reacts directly on a sulfur atom instead of abstracting a hydrogen atom. Using theoretical calculations based on our high-resolution structures we followed the evolution of the electronic structure from SAM through to the formation of S-adenosyl-L-cysteine. Our results suggest that, at least in this case, the widely proposed and highly reactive 5'-deoxyadenosyl radical species that triggers the reaction in radical SAM enzymes is not an isolable intermediate.

The radical S-adenosyl-L-methionine tryptophan lyase NosL converts L-tryptophan into 3-methylindolic acid, which is a precursor in the synthesis of the thiopeptide antibiotic nosiheptide. Using electron paramagnetic resonance spectroscopy and multiple L-tryptophan isotopologues, we trapped and characterized radical intermediates that indicate a carboxyl fragment migration mechanism for NosL. This is in contrast to a proposed fragmentation-recombination mechanism that implied Cα-Cβ bond cleavage of L-tryptophan. Although NosL resembles related tyrosine lyases, subtle substrate motions in its active site are responsible for a fine-tuned radical chemistry, which selects the Cα-C bond for disruption. This mechanism highlights evolutionary adaptation to structural constraints in proteins as a route to alternative enzyme function., (Copyright © 2016, American Association for the Advancement of Science.)

The synthesis and assembly of the active site [FeFe] unit of [FeFe]-hydrogenases require at least three maturases. The radical S-adenosyl-l-methionine HydG, the best characterized of these proteins, is responsible for the synthesis of the hydrogenase CO and CN(-) ligands from tyrosine-derived dehydroglycine (DHG). We speculated that CN(-) and the CO precursor (-):CO2H may be generated through an elimination reaction. We tested this hypothesis with both wild type and HydG variants defective in second iron-sulfur cluster coordination by measuring the in vitro production of CO, CN(-), and (-):CO2H-derived formate. We indeed observed formate production under these conditions. We conclude that HydG is a multifunctional enzyme that produces DHG, CN(-), and CO at three well-differentiated catalytic sites. We also speculate that homocysteine, cysteine, or a related ligand could be involved in Fe(CO)x(CN)y transfer to the HydF carrier/scaffold.

The structure of the dimeric holo-fumarate and nitrate reduction regulator (FNR) from Aliivibrio fischeri has been solved at 2.65 Å resolution. FNR globally controls the transition between anaerobic and aerobic respiration in facultative anaerobes through the assembly/degradation of its oxygen-sensitive [4Fe-4S] cluster. In the absence of O2, FNR forms a dimer and specifically binds to DNA, whereas in its presence, the cluster is degraded causing FNR monomerization and DNA dissociation. We have used our crystal structure and the information previously gathered from numerous FNR variants to propose that this process is governed by extremely fine-tuned interactions, mediated by two salt bridges near the amino-terminal cluster-binding domain and an "imperfect" coiled-coil dimer interface. [4Fe-4S] to [2Fe-2S] cluster degradation propagates a conformational signal that indirectly causes monomerization by disrupting the first of these interactions and unleashing the "unzipping" of the FNR dimer in the direction of the carboxyl-terminal DNA binding domain.

NosL is a member of a family of radical S-adenosylmethionine enzymes that catalyze the cleavage of the Cα-Cβ bond of aromatic amino acids. In this paper, we describe a set of experiments with substrate analogues and mutants for probing the early steps of the NosL mechanism. We provide biochemical evidence in support of the structural studies showing that the 5'-deoxyadenosyl radical abstracts a hydrogen atom from the amino group of tryptophan. We demonstrate that d-tryptophan is a substrate for NosL but shows relaxed regio control of the first β-scission reaction. Mutagenesis studies confirm that Arg323 is important for controlling the regiochemistry of the β-scission reaction and that Ser340 binds the substrate by hydrogen bonding to the indolic N1 atom.

In natural photosynthesis, light is used for the production of chemical energy carriers to fuel biological activity. The re-engineering of natural photosynthetic pathways can provide inspiration for sustainable fuel production and insights for understanding the process itself. Here, we employ a semiartificial approach to study photobiological water splitting via a pathway unavailable to nature: the direct coupling of the water oxidation enzyme, photosystem II, to the H2 evolving enzyme, hydrogenase. Essential to this approach is the integration of the isolated enzymes into the artificial circuit of a photoelectrochemical cell. We therefore developed a tailor-made hierarchically structured indium-tin oxide electrode that gives rise to the excellent integration of both photosystem II and hydrogenase for performing the anodic and cathodic half-reactions, respectively. When connected together with the aid of an applied bias, the semiartificial cell demonstrated quantitative electron flow from photosystem II to the hydrogenase with the production of H2 and O2 being in the expected two-to-one ratio and a light-to-hydrogen conversion efficiency of 5.4% under low-intensity red-light irradiation. We thereby demonstrate efficient light-driven water splitting using a pathway inaccessible to biology and report on a widely applicable in vitro platform for the controlled coupling of enzymatic redox processes to meaningfully study photocatalytic reactions.

Iron sulfur ([Fe-S]) clusters are essential prosthetic groups involved in fundamental cell processes such as gene expression regulation, electron transfer and Lewis acid base chemistry. Central components of their biogenesis are pyridoxal-5'-phosphate (PLP) dependent l-cysteine desulfurases, which provide the necessary S atoms for [Fe-S] cluster assembly. The archaeon Archaeoglobus fulgidus (Af) has two ORFs, which although annotated as l-cysteine desulfurases of the ISC type (IscS), lack the essential Lys residue (K199 in Af) that forms a Schiff base with PLP. We have previously determined the structure of an Af(IscU-D35A-IscS)2 complex heterologously expressed in Escherichia coli and found it to contain a [Fe2S2] cluster. In order to understand the origin of sulfide in that structure we have performed a series of functional tests using wild type and mutated forms of AfIscS. In addition, we have determined the crystal structure of an AfIscS-D199K mutant. From these studies we conclude that: i) AfIscS has no desulfurase activity; ii) in our in vitro [Fe2S2] cluster assembly experiments, sulfide ions are non-enzymatically generated by a mixture of iron, l-cysteine and PLP and iii) the physiological role of AfIscS may be to provide a cysteine ligand to the nascent cluster as observed in the [Fe2S2]-Af(IscU-D35A-IscS)2 complex. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

[NiFe]-hydrogenases are well-studied enzymes capable of oxidizing molecular hydrogen and reducing protons. EPR and FTIR spectroscopic studies have shown that these enzymes can be isolated in several redox states that include paramagnetic oxidized inactive Ni-A and Ni-B species and a reduced Ni-C form. The latter and the diamagnetic respectively more oxidized Ni-SI and more reduced Ni-R forms are generally thought to be involved in the catalytic cycle of [NiFe]-hydrogenases. With the exception of Ni-SI, these different stable states have been well characterized. Here, based on the crystal structure of a partially reduced Desulfovibrio fructosovorans (Df) enzyme and data from the literature we propose that at least one of the Ni-SI sub-states contains an unexpected combination of hydride and sulfenic acid moieties. We have also determined the structure of the less oxygen-sensitive Df [NiFe]-hydrogenase V74C mutant and found that more than half of the active site nickel occupies a novel position, called Ni'. In this new position, the metal ion is coordinated by two cysteine thiolates, a bridging species modeled as SH(-) and a main chain carboxamido N atom. The Ni' coordination is similar to the one found in Ni superoxide dismutase, an enzyme that operates at significantly more positive potentials than [NiFe]-hydrogenases. We propose that the oxygen-tolerance of the V74C variant results from a high potential stabilization of a Ni'(iii) species induced by the change in the metal ion coordination sphere. We also propose that transient Ni'(iii) species can rapidly attract successive electrons from the Fe4S4 proximal cluster accelerating the reduction of oxygen to water and hydroxide. The naturally occurring oxygen-tolerant [NiFe]-hydrogenases have an unusual proximal cluster that has been shown to be exceptionally plastic and capable of undergoing two successive one-electron oxidations. This double oxidation is modulated by the migration of one of the iron atoms in the cluster to the main chain where, as Fe(iii), it forms a bond with a carboxamido N ligand. Like in the Df V74C variant the electrons from the proximal cluster help reducing O2 to H2O and OH(-). In conclusion, in both cases a metal-carboxamido bond may explain, at least partially, the observed oxygen tolerance.

The structure of the radical S-adenosyl-L-methionine (SAM) [FeFe]-hydrogenase maturase HydG involved in CN(-) /CO synthesis is characterized by two internal tunnels connecting its tyrosine-binding pocket with the external medium and the C-terminal Fe4 S4 cluster-containing region. A comparison with a tryptophan-bound NosL structure suggests that substrate binding causes the closing of the first tunnel and, along with mutagenesis studies, that tyrosine binds to HydG with its amino group well positioned for H-abstraction by SAM. In this orientation the dehydroglycine (DHG) fragment caused by tyrosine Cα-Cβ bond scission can readily migrate through the second tunnel towards the C-terminal domain where both CN(-) and CO are synthesized. Our HydG structure appears to be in a relaxed state with its C-terminal cluster CysX2 CysX22 Cys motif exposed to solvent. A rotation of this domain coupled to Fe4 S4 cluster assembly would bury its putatively reactive unique Fe ion thereby allowing it to interact with DHG., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-'Sox' species.

Spark-tube experiments and analysis of meteorite contents have led to the widespread notion that abiotic organic molecules were the first life components. However, there is a contradiction between the abundance of simple molecules, such as the amino acids glycine and alanine, observed in these studies, and the minimal functional complexity that even the least sophisticated living system should require. I will argue that although simple abiotic molecules must have primed proto-metabolic pathways, only Darwinian evolving systems could have generated life. This condition may have been initially fulfilled by both replicating RNAs and autocatalytic reaction chains, such as the reductive citric acid cycle. The interactions between nucleotides and biotic amino acids, which conferred new functionalities to the former, also resulted in the progressive stereochemical recognition of the latter by cognate anticodons. At this point only large enough amino acids would be recognized by the primordial RNA adaptors and could polymerize forming the first peptides. The gene duplication of RNA adaptors was a crucial event. By removing one of the anticodons from the acceptor stem the new RNA adaptor liberated itself from the stereochemical constraint and could be acylated by smaller amino acids. The emergence of messenger RNA and codon capture followed.

Streptomyces actuosus tryptophan lyase (NosL) is a radical SAM enzyme which catalyzes the synthesis of 3-methyl-2-indolic acid, a precursor in the synthesis of the promising antibiotic nosiheptide. The reaction involves cleavage of the tryptophan Cα-Cβ bond and recombination of the amino-acid-derived -COOH fragment at the indole ring. Reported herein is the 1.8 Å resolution crystal structure of NosL complexed with its substrate. Unexpectedly, only one of the tryptophan amino hydrogen atoms is optimally placed for H abstraction by the SAM-derived 5'-deoxyadenosyl radical. This orientation, in turn, rules out the previously proposed delocalized indole radical as the species which undergoes Cα-Cβ bond cleavage. Instead, stereochemical considerations indicate that the reactive intermediate is a (·)NH tryptophanyl radical. A structure-based amino acid sequence comparison of NosL with the tyrosine lyases ThiH and HydG strongly suggests that an equivalent (·)NH radical operates in the latter enzymes., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of diimines derived from saturated and unsaturated fatty acids and including a dipyridylamine (dpa) or a bispyridylmethane (bpm) scaffold as a chelating moiety were synthesized and characterized spectroscopically. Complexation by [LM(μ-Cl)Cl]2 (M = Ru, L = p-cymene; M = Rh, L = Cp*) afforded the monocationic, mononuclear complexes of general formula [LM(N^N)Cl]Cl with N^N being the diimine ligand. Unsurprisingly, these new complexes catalysed the transfer hydrogenation of an activated aromatic ketone, namely 2,2,2-trifluoroacetophenone (TFACP), in water at neutral pH and mild temperature in the presence of formate as a hydrogen donor. The catalytic activity of the complexes expressed as TOF was shown to depend not only on the metal (Ru or Rh) but also on the chelating entity (dpa or bpm) and the length and nature of the lipidic chain tethered to it. Incorporation of the complexes within bovine β-lactoglobulin (βLG) as the protein host was studied by circular dichroism and fluorescence spectroscopy and again noticeable differences were observed between the saturated and unsaturated fatty acid derivatives. Eventually, the ability of the protein hybrids to catalyse the transfer hydrogenation of TFACP was demonstrated. Good-to-quantitative conversions in the corresponding alcohol were reached within 72 h with the rhodium(iii) hybrids and the best enantioselectivities (up to 32% ee for the (R)-enantiomer) were measured with the Rh(iii) cofactors derived from palmitic and stearic acids once incorporated into the isoform A of βLG.

Quinolinate synthase (NadA) is a Fe4S4 cluster-containing dehydrating enzyme involved in the synthesis of quinolinic acid (QA), the universal precursor of the essential nicotinamide adenine dinucleotide (NAD) coenzyme. A previously determined apo NadA crystal structure revealed the binding of one substrate analog, providing partial mechanistic information. Here, we report on the holo X-ray structure of NadA. The presence of the Fe4S4 cluster generates an internal tunnel and a cavity in which we have docked the last precursor to be dehydrated to form QA. We find that the only suitably placed residue to initiate this process is the conserved Tyr21. Furthermore, Tyr21 is close to a conserved Thr-His-Glu triad reminiscent of those found in proteases and other hydrolases. Our mutagenesis data show that all of these residues are essential for activity and strongly suggest that Tyr21 deprotonation, to form the reactive nucleophilic phenoxide anion, is mediated by the triad. NadA displays a dehydration mechanism significantly different from the one found in archetypical dehydratases such as aconitase, which use a serine residue deprotonated by an oxyanion hole. The X-ray structure of NadA will help us unveil its catalytic mechanism, the last step in the understanding of NAD biosynthesis.

The most efficient catalysts for solar fuel production should operate close to reversible potentials, yet possess a bias for the fuel-forming direction. Protein film electrochemical studies of Ni-containing carbon monoxide dehydrogenase and [NiFeSe]-hydrogenase, each a reversible electrocatalyst, show that the electronic state of the electrode strongly biases the direction of electrocatalysis of CO2/CO and H(+)/H2 interconversions. Attached to graphite electrodes, these enzymes show high activities for both oxidation and reduction, but there is a marked shift in bias, in favor of CO2 or H(+) reduction, when the respective enzymes are attached instead to n-type semiconductor electrodes constructed from CdS and TiO2 nanoparticles. This catalytic rectification effect can arise for a reversible electrocatalyst attached to a semiconductor electrode if the electrode transforms between semiconductor- and metallic-like behavior across the same narrow potential range (<0.25 V) that the electrocatalytic current switches between oxidation and reduction.

This study shows how the NiFeSe site of an anaerobically purified O2-resistant hydrogenase reacts with air to give a seleninate as the first product. Less oxidized states of the active site are readily reduced in the presence of X-rays. Reductive enzyme activation requires an efficient pathway for water escape.

Fe4S4 clusters are very common versatile prosthetic groups in proteins. Their redox property of being sensitive to O2-induced oxidative damage is, for instance, used by the cell to sense oxygen levels and switch between aerobic and anaerobic metabolisms, as exemplified by the fumarate, nitrate reduction regulator (FNR). Using the hydrogenase maturase HydE from Thermotoga maritima as a template, we obtained several unusual forms of FeS clusters, some of which are associated with important structural changes. These structures represent intermediate states relevant to both FeS cluster assembly and degradation. We observe one Fe2S2 cluster bound by two cysteine persulfide residues. This observation lends structural support to a very recent Raman study, which reported that Fe4S4-to-Fe2S2 cluster conversion upon oxygen exposure in FNR resulted in concomitant production of cysteine persulfide as cluster ligands. Similar persulfide ligands have been observed in vitro for several other Fe4S4 cluster-containing proteins. We have also monitored FeS cluster conversion directly in our protein crystals. Our structures indicate that the Fe4S4-to-Fe2S2 change requires large structural modifications, which are most likely responsible for the dimer-monomer transition in FNR.

The ABC-type importer NikABCDE mediates nickel acquisition in Escherichia coli. The periplasmic nickel-binding component NikA has a folding similar to that of the peptide transporter OppA and does not bind free nickel. Instead, we showed that the metal is tetra-coordinated by an organic tri-dentate molecule and His416. Conversely, it has been recently reported that NikA binds Ni-(L-His)2 and that addition of histidine increases the rate of nickel uptake in vivo. Here, we report the structure of NikA/Ni-(L-His)2 and show that histidine binding differs from peptide binding in OppA. The structure also confirms the central role of His416 in nickel binding., (Copyright © 2013 Elsevier Inc. All rights reserved.)

L-Cysteine desulfurase IscS and scaffold IscU proteins are universally involved in Fe/S cluster synthesis. The Archaeoglobus fulgidus (Af) genome encodes proteins having a high degree of primary structure similarity to IscS and IscU from other organisms. However, AfIscS is unusual because it lacks the active site lysine residue that normally forms an internal Schiff base with pyridoxal-phosphate (PLP) and serves as a base during catalysis. Our as-isolated recombinant AfIscS contains pyridoxamine phosphate (PMP) instead of the expected PLP and lacks desulfurase activity. We have solved its structure to 1.43 Å resolution and found that PMP binds non-covalently at the PLP site of the enzyme and displays significant disorder. However, the previously reported structure of recombinant Af(IscU-D35A-IscS)(2) contains an in vivo generated [Fe(2)S(2)] species within AfIscU and the question arises as to how its sulfides were generated. Here, we report that adding PLP to AfIscS produces an enzyme that displays in vitro L-cysteine desulfurase activity mediating the synthesis of a stable holo Af(IscU-D35A-IscS) complex.

"Hyd-1", produced by Escherichia coli , exemplifies a special class of [NiFe]-hydrogenase that can sustain high catalytic H(2) oxidation activity in the presence of O(2)-an intruder that normally incapacitates the sulfur- and electron-rich active site. The mechanism of "O(2) tolerance" involves a critical role for the Fe-S clusters of the electron relay, which is to ensure the availability-for immediate transfer back to the active site-of all of the electrons required to reduce an attacking O(2) molecule completely to harmless H(2)O. The unique [4Fe-3S] cluster proximal to the active site is crucial because it can rapidly transfer two of the electrons needed. Here we investigate and establish the equally crucial role of the high potential medial [3Fe-4S] cluster, located >20 Å from the active site. A variant, P242C, in which the medial [3Fe-4S] cluster is replaced by a [4Fe-4S] cluster, is unable to sustain steady-state H(2) oxidation activity in 1% O(2). The [3Fe-4S] cluster is essential only for the first stage of complete O(2) reduction, ensuring the supply of all three electrons needed to form the oxidized inactive state "Ni-B" or "Ready" (Ni(III)-OH). Potentiometric titrations show that Ni-B is easily reduced (E(m) ≈ +0.1 V at pH 6.0); this final stage of the O(2)-tolerance mechanism regenerates active enzyme, effectively completing a competitive four-electron oxidase cycle and is fast regardless of alterations at the proximal or medial clusters. As a consequence of all these factors, the enzyme's response to O(2), viewed by its electrocatalytic activity in protein film electrochemistry (PFE) experiments, is merely to exhibit attenuated steady-state H(2) oxidation activity; thus, O(2) behaves like a reversible inhibitor rather than an agent that effectively causes irreversible inactivation. The data consolidate a rich picture of the versatile role of Fe-S clusters in electron relays and suggest that Hyd-1 can function as a proficient hydrogen oxidase.