Eberhard Warkentin

The crystal structure of [Fe]-hydrogenase reveals the geometry of the active site.

Biological formation and consumption of molecular hydrogen (H2) are catalyzed by hydrogenases, of which three phylogenetically unrelated types are known: [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-hydrogenase. We present a crystal structure of [Fe]-hydrogenase at 1.75 angstrom resolution, showing a mononuclear iron coordinated by the sulfur of cysteine 176, two carbon monoxide (CO) molecules, and the sp2-hybridized nitrogen of a 2-pyridinol compound with back-bonding properties similar to those of cyanide. The three-dimensional arrangement of the ligands is similar to that of thiolate, CO, and cyanide ligated to the low-spin iron in binuclear [NiFe]- and [FeFe]-hydrogenases, although the enzymes have evolved independently and the CO and cyanide ligands are not found in any other metalloenzyme. The related iron ligation pattern of hydrogenases exemplifies convergent evolution and presumably plays an essential role in H2 activation. This finding may stimulate the ongoing synthesis of catalysts that could substitute for platinum in applications such as fuel cells.

The Structure of cbb3 Cytochrome Oxidase Provides Insights into Proton Pumping

C-Family Oxidase Structure The heme-copper oxidases (HCOs) play a key role in aerobic respiration by coupling oxygen reduction to transmembrane proton pumping to generate electrochemical ion gradients across biological membranes that provide energy for many cellular processes. Based on subunit composition, electron donor, and heme type, the HCOs are divided into three families, of which families A and B have been structurally characterized. Buschmann et al. (p. 327, published online 24 June) now report the crystal structure of the C-family cbb3 oxidase from Pseudomonas stutzeri. The structure suggests a different redox-driven pumping mechanism from A and B HCOs, and gives insight into why C HCOs are catalytically active at low oxygen concentrations. The third class of heme-copper oxidases reveals new structural solutions to achieve redox-driven proton translocation. The heme-copper oxidases (HCOs) accomplish the key event of aerobic respiration; they couple O2 reduction and transmembrane proton pumping. To gain new insights into the still enigmatic process, we structurally characterized a C-family HCO—essential for the pathogenicity of many bacteria—that differs from the two other HCO families, A and B, that have been structurally analyzed. The x-ray structure of the C-family cbb3 oxidase from Pseudomonas stutzeri at 3.2 angstrom resolution shows an electron supply system different from families A and B. Like family-B HCOs, C HCOs have only one pathway, which conducts protons via an alternative tyrosine-histidine cross-link. Structural differences around hemes b and b3 suggest a different redox-driven proton-pumping mechanism and provide clues to explain the higher activity of family-C HCOs at low oxygen concentrations.

Insight into the mechanism of biological methanol activation based on the crystal structure of the methanol-cobalamin methyltransferase complex

Some methanogenic and acetogenic microorganisms have the catalytic capability to cleave heterolytically the CO bond of methanol. To obtain insight into the elusive enzymatic mechanism of this challenging chemical reaction we have investigated the methanol-activating MtaBC complex from Methanosarcina barkeri composed of the zinc-containing MtaB and the 5-hydroxybenzimidazolylcobamide-carrying MtaC subunits. Here we report the 2.5-Å crystal structure of this complex organized as a (MtaBC)2 heterotetramer. MtaB folds as a TIM barrel and contains a novel zinc-binding motif. Zinc(II) lies at the bottom of a funnel formed at the C-terminal β-barrel end and ligates to two cysteinyl sulfurs (Cys-220 and Cys-269) and one carboxylate oxygen (Glu-164). MtaC is structurally related to the cobalamin-binding domain of methionine synthase. Its corrinoid cofactor at the top of the Rossmann domain reaches deeply into the funnel of MtaB, defining a region between zinc(II) and the corrinoid cobalt that must be the binding site for methanol. The active site geometry supports a SN2 reaction mechanism, in which the CO bond in methanol is activated by the strong electrophile zinc(II) and cleaved because of an attack of the supernucleophile cob(I)amide. The environment of zinc(II) is characterized by an acidic cluster that increases the charge density on the zinc(II), polarizes methanol, and disfavors deprotonation of the methanol hydroxyl group. Implications of the MtaBC structure for the second step of the reaction, in which the methyl group is transferred to coenzyme M, are discussed.

Structure of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, the terminal enzyme of the non-mevalonate pathway.

Molecular evolution has evolved two metabolic routes for isoprenoid biosynthesis: the mevalonate and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The MEP pathway is used by most pathogenic bacteria and some parasitic protozoa (including the malaria parasite, Plasmodium falciparum) as well as by plants, but is not present in animals. The terminal reaction of the MEP pathway is catalyzed by (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase (LytB), an enzyme that converts HMBPP into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Here, we present the structure of Aquifex aeolicus LytB, at 1.65 A resolution. The protein adopts a cloverleaf or trefoil-like structure with each monomer in the dimer containing three alpha/beta domains surrounding a central [Fe3S4] cluster ligated to Cys13, Cys96, and Cys193. Two highly conserved His (His 42 and His 124) and a totally conserved Glu (Glu126) are located in the same central site and are proposed to be involved in ligand binding and catalysis. Substrate access is proposed to occur from the front-side face of the protein, with the HMBPP diphosphate binding to the two His and the 4OH of HMBPP binding to the fourth iron thought to be present in activated clusters, while Glu126 provides the protons required for IPP/DMAPP formation.

Structure and function of enzymes involved in the methanogenic pathway utilizing carbon dioxide and molecular hydrogen

Methane is an end product of anaerobic degradation of organic compounds in fresh water environments such as lake sediments and the intestinal tract of animals. Methanogenic archaea produce methane from carbon dioxide and molecular hydrogen, acetate and C1 compounds such as methanol in an energy gaining process. The methanogenic pathway utilizing carbon dioxide and molecular hydrogen involves ten methanogen specific enzymes, which catalyze unique reactions using novel coenzymes. These enzymes have been purified and biochemically characterized. The genes encoding the enzymes have been cloned and sequenced. Recently, crystal structures of five methanogenic enzymes: formylmethanofuran : tetrahydromethanopterin formyltransferase, methenyltetrahydromethanopterin cyclohydrolase, methylenetetrahydromethanopterin reductase, F420H2:NADP oxidoreductase and methyl-coenzyme M reductase were reported. In this review, we describe the pathway utilizing carbon dioxide and molecular hydrogen and the catalytic mechanisms of the enzymes based on their crystal structures.

Structure of the Dissimilatory Sulfite Reductase from the Hyperthermophilic Archaeon Archaeoglobus fulgidus

Conservation of energy based on the reduction of sulfate is of fundamental importance for the biogeochemical sulfur cycle. A key enzyme of this ancient anaerobic process is the dissimilatory sulfite reductase (dSir), which catalyzes the six-electron reduction of sulfite to hydrogen sulfide under participation of a unique magnetically coupled siroheme-[4Fe-4S] center. We determined the crystal structure of the enzyme from the sulfate-reducing archaeon Archaeoglobus fulgidus at 2-A resolution and compared it with that of the phylogenetically related assimilatory Sir (aSir). dSir is organized as a heterotetrameric (alphabeta)(2) complex composed of two catalytically independent alphabeta heterodimers. In contrast, aSir is a monomeric protein built of two fused modules that are structurally related to subunits alpha and beta except for a ferredoxin domain inserted only into the subunits of dSir. The [4Fe-4S] cluster of this ferredoxin domain is considered as the terminal redox site of the electron transfer pathway to the siroheme-[4Fe-4S] center in dSir. While aSir binds one siroheme-[4Fe-4S] center, dSir harbors two of them within each alphabeta heterodimer. Surprisingly, only one siroheme-[4Fe-4S] center in each alphabeta heterodimer is catalytically active, whereas access to the second one is blocked by a tryptophan residue. The spatial proximity of the functional and structural siroheme-[4Fe-4S] centers suggests that the catalytic activity at one active site was optimized during evolution at the expense of the enzymatic competence of the other. The sulfite binding mode and presumably the mechanism of sulfite reduction appear to be largely conserved between dSir and aSir. In addition, a scenario for the evolution of Sirs is proposed.

Structure of coenzyme F420H2 oxidase (FprA), a di‐iron flavoprotein from methanogenic Archaea catalyzing the reduction of O2 to H2O

The di‐iron flavoprotein F420H2 oxidase found in methanogenic Archaea catalyzes the four‐electron reduction of O2 to 2H2O with 2 mol of reduced coenzyme F420(7,8‐dimethyl‐8‐hydroxy‐5‐deazariboflavin). We report here on crystal structures of the homotetrameric F420H2 oxidase from Methanothermobacter marburgensis at resolutions of 2.25 Å, 2.25 Å and 1.7 Å, respectively, from which an active reduced state, an inactive oxidized state and an active oxidized state could be extracted. As found in structurally related A‐type flavoproteins, the active site is formed at the dimer interface, where the di‐iron center of one monomer is juxtaposed to FMN of the other. In the active reduced state [Fe(II)Fe(II)FMNH2], the two irons are surrounded by four histidines, one aspartate, one glutamate and one bridging aspartate. The so‐called switch loop is in a closed conformation, thus preventing F420 binding. In the inactive oxidized state [Fe(III)FMN], the iron nearest to FMN has moved to two remote binding sites, and the switch loop is changed to an open conformation. In the active oxidized state [Fe(III)Fe(III)FMN], both irons are positioned as in the reduced state but the switch loop is found in the open conformation as in the inactive oxidized state. It is proposed that the redox‐dependent conformational change of the switch loop ensures alternate complete four‐electron O2 reduction and redox center re‐reduction. On the basis of the known Si–Si stereospecific hydride transfer, F420H2 was modeled into the solvent‐accessible pocket in front of FMN. The inactive oxidized state might provide the molecular basis for enzyme inactivation by long‐term O2 exposure observed in some members of the FprA family.

Structures of F420H2:NADP+ oxidoreductase with and without its substrates bound.

Cofactor F420 is a 5′‐deazaflavin derivative first discovered in methanogenic archaea but later found also to be present in some bacteria. As a coenzyme, it is involved in hydride transfer reactions and as a prosthetic group in the DNA photolyase reaction. We report here for the first time on the crystal structure of an F420‐dependent oxidoreductase bound with F420. The structure of F420H2:NADP+ oxidoreductase resolved to 1.65 Å contains two domains: an N‐terminal domain characteristic of a dinucleotide‐binding Rossmann fold and a smaller C‐terminal domain. The nicotinamide and the deazaflavin part of the two coenzymes are bound in the cleft between the domains such that the Si‐faces of both face each other at a distance of 3.1 Å, which is optimal for hydride transfer. Comparison of the structures bound with and without substrates reveals that of the two substrates NADP has to bind first, the binding being associated with an induced fit.

Reaction Cycle of the Dissimilatory Sulfite Reductase from Archaeoglobus fulgidus

A vital process in the biogeochemical sulfur cycle is the dissimilatory sulfate reduction pathway in which sulfate (SO₄⁻²) is converted to hydrogen sulfide (H₂S). Dissimilatory sulfite reductase (dSir), its key enzyme, hosts a unique siroheme-[4Fe-4S] cofactor and catalyzes the six-electron reduction of sulfite (SO₃²⁻) to H₂S. To explore this reaction, we determined the X-ray structures of dSir from the archaeon Archaeoglobus fulgidus in complex with sulfite, sulfide (S²⁻) carbon monoxide (CO), cyanide (CN⁻), nitrite (NO₂⁻), nitrate (NO₃⁻), and phosphate (PO₄³⁻). Activity measurements indicated that dSir of A. fulgidus reduces, besides sulfite and nitrite, thiosulfate (S₂O₃²⁻) and trithionate (S₃O₆²⁻) and produces the latter two compounds besides sulfide. On this basis, a three-step mechanism was proposed, each step consisting of a two-electron transfer, a two-proton uptake, and a dehydration event. In comparison, the related active site structures of the assimilatory sulfite reductase (aSir)- and dSir-SO₃²⁻complexes reveal different conformations of Argα170 and Lysα211 both interacting with the sulfite oxygens (its sulfur atom coordinates the siroheme iron), a sulfite rotation of ~60° relative to each other, and different access of solvent molecules to the sulfite oxygens from the active site cleft. Therefore, solely in dSir a further sulfite molecule can be placed in van der Waals contact with the siroheme-ligated sulfite or sulfur-oxygen intermediates necessary for forming thiosulfate and trithionate. Although reported for dSir from several sulfate-reducing bacteria, the in vivo relevance of their formation is questionable.

Crystal structure of methylenetetrahydromethanopterin reductase (Mer) in complex with coenzyme F420: Architecture of the F420/FMN binding site of enzymes within the nonprolyl cis‐peptide containing bacterial luciferase family

Methylenetetratetrahydromethanopterin reductase (Mer) is involved in CO2 reduction to methane in methanogenic archaea and catalyses the reversible reduction of methylenetetrahydromethanopterin (methylene‐H4MPT) to methyl‐H4MPT with coenzyme F420H2, which is a reduced 5′‐deazaflavin. Mer was recently established as a TIM barrel structure containing a nonprolyl cis‐peptide bond but the binding site of the substrates remained elusive. We report here on the crystal structure of Mer in complex with F420 at 2.6 Å resolution. The isoalloxazine ring is present in a pronounced butterfly conformation, being induced from the Re‐face of F420 by a bulge that contains the non‐prolyl cis‐peptide bond. The bindingmode of F420 is very similar to that in F420‐dependent alcohol dehydrogenase Adf despite the low sequence identity of 21%. Moreover, binding of F420 to the apoenzyme was only associated with minor conformational changes of the polypeptide chain. These findings allowed us to build an improved model of FMN into its binding site in bacterial luciferase, which belongs to the same structural family as Mer and Adf and also contains a nonprolyl cis‐peptide bond in an equivalent position.