The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb 3-Type Cytochrome Oxidase Biogenesis.

Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb 3-type cytochrome oxidase (cbb 3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb 3-Cox. CcoI and cbb 3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb 3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb 3-Cox assembly.

The Cofactors of Nitrogenases.

In biological nitrogen fixation, the enzyme nitrogenase mediates the reductive cleavage of the stable triple bond of gaseous N2at ambient conditions, driven by the hydrolysis of ATP, to yield bioavailable ammonium (NH4+). At the core of nitrogenase is a complex, ironsulfur based cofactor that in most variants of the enzyme contains an additional, apical heterometal (Mo or V), an organic homocitrate ligand coordinated to this heterometal, and a unique, interstitial carbide. Recent years have witnessed fundamental advances in our understanding of the atomic and electronic structure of the nitrogenase cofactor. Spectroscopic studies have succeeded in trapping and identifying reaction intermediates and several inhibitor- or intermediate- bound structures of the cofactors were characterized by high-resolution X-ray crystallography. Here we summarize the current state of understanding of the cofactors of the nitrogenase enzymes, their interplay in electron transfer and in the six-electron reduction of nitrogen to ammonium and the actual theoretical and experimental conclusion on how this challenging chemistry is achieved.

The Critical E4 State of Nitrogenase Catalysis.

The reaction catalyzed by the nitrogenase enzyme involves breaking the stable triple bond of the dinitrogen molecule and is consequently considered among the most challenging reactions in biology. While many aspects regarding its atomic mechanism remain to be elucidated, a kinetic scheme established by David Lowe and Roger Thorneley has remained a gold standard for functional studies of the enzyme for more than 30 years. Recent three-dimensional structures of ligand-bound states of molybdenum- and vanadium-dependent nitrogenases have revealed the actual site of substrate binding on the large active site cofactors of this class of enzymes. The binding mode of an inhibitor and a reaction intermediate further substantiate a hypothesis by Seefeldt, Hoffman, and Dean that the activation of N2 is made possible by a reductive elimination of H2 that leaves the cofactor in a super-reduced state that can bind and reduce the inert N2 molecule. Here we discuss the immediate implications of the structurally observed mode of binding of small molecules to the enzyme with respect to the early stages of the Thorneley-Lowe mechanism of nitrogenase. Four consecutive single-electron reductions give rise to two bridging hydrides at the cluster surface that can recombine to eliminate H2 and enable the reduced cluster to bind its substrate in a bridging mode.

A bound reaction intermediate sheds light on the mechanism of nitrogenase.

Reduction of N2 by nitrogenases occurs at an organometallic iron cofactor that commonly also contains either molybdenum or vanadium. The well-characterized resting state of the cofactor does not bind substrate, so its mode of action remains enigmatic. Carbon monoxide was recently found to replace a bridging sulfide, but the mechanistic relevance was unclear. Here we report the structural analysis of vanadium nitrogenase with a bound intermediate, interpreted as a µ2-bridging, protonated nitrogen that implies the site and mode of substrate binding to the cofactor. Binding results in a flip of amino acid glutamine 176, which hydrogen-bonds the ligand and creates a holding position for the displaced sulfide. The intermediate likely represents state E6 or E7 of the Thorneley-Lowe model and provides clues to the remainder of the catalytic cycle.

Signaling ammonium across membranes through an ammonium sensor histidine kinase.

Sensing and uptake of external ammonium is essential for anaerobic ammonium-oxidizing (anammox) bacteria, and is typically the domain of the ubiquitous Amt/Rh ammonium transporters. Here, we report on the structure and function of an ammonium sensor/transducer from the anammox bacterium "Candidatus Kuenenia stuttgartiensis" that combines a membrane-integral ammonium transporter domain with a fused histidine kinase. It contains a high-affinity ammonium binding site not present in assimilatory Amt proteins. The levels of phosphorylated histidine in the kinase are coupled to the presence of ammonium, as conformational changes during signal recognition by the Amt module are transduced internally to modulate the kinase activity. The structural analysis of this ammonium sensor by X-ray crystallography and small-angle X-ray-scattering reveals a flexible, bipartite system that recruits a large uptake transporter as a sensory module and modulates its functionality to achieve a mechanistic coupling to a kinase domain in order to trigger downstream signaling events.

Secondary Structure Determination by Means of ATR-FTIR Spectroscopy.

Specialized infrared spectroscopic techniques have been developed that allow studying the secondary structure of membrane proteins and the influence of crucial parameters like lipid content and detergent. Here, we focus on an ATR-FTIR spectroscopic study of Af-Amt1 and the influence of LDAO/glycerol on its structural integrity. Our results clearly indicate that infrared spectroscopy can be used to identify the adapted sample conditions.

The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction.

The copper-containing enzyme nitrous oxide reductase (N2OR) catalyzes the transformation of nitrous oxide (N2O) to dinitrogen (N2) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites CuA and CuZ, and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes N2O respiration. Here we demonstrate through biochemical and structural studies that the ApbE protein from Pseudomonas stutzeri, where the nosX gene is absent, is a monomeric FAD-binding protein that can serve as the flavin donor for NosR maturation via covalent flavinylation of a threonine residue. The flavin transfer reaction proceeds both in vivo and in vitro to generate post-translationally modified NosR with covalently bound FMN. Only FAD can act as substrate and the reaction requires a divalent cation, preferably Mg2+ that was also present in the crystal structure. In addition, the reaction is species-specific to a certain extent.

Production and isolation of vanadium nitrogenase from Azotobacter vinelandii by molybdenum depletion.

The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N2 under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfD2K2G2 heterohexamer.

Nitrogenase FeMoco investigated by spatially resolved anomalous dispersion refinement.

The [Mo:7Fe:9S:C] iron-molybdenum cofactor (FeMoco) of nitrogenase is the largest known metal cluster and catalyses the 6-electron reduction of dinitrogen to ammonium in biological nitrogen fixation. Only recently its atomic structure was clarified, while its reactivity and electronic structure remain under debate. Here we show that for its resting S=3/2 state the common iron oxidation state assignments must be reconsidered. By a spatially resolved refinement of the anomalous scattering contributions of the 7 Fe atoms of FeMoco, we conclude that three irons (Fe1/3/7) are more reduced than the other four (Fe2/4/5/6). Our data are in agreement with the recently revised oxidation state assignment for the molybdenum ion, providing the first spatially resolved picture of the resting-state electron distribution within FeMoco. This might provide the long-sought experimental basis for a generally accepted theoretical description of the cluster that is in line with available spectroscopic and functional data.

Active sites without restraints: high-resolution analysis of metal cofactors.

For most three-dimensional structures of biological macromolecules, the factual accuracy of atom positions by far exceeds the resolution of the experimental data, although the refinement problem presented by a protein structure is substantially underdetermined. This is achieved through using restraints that precisely define protein geometries and thus reduce the degrees of freedom of the refinement problem. If such information is not available or when unusual geometries or particular ligand states complicate structural analysis, possible pitfalls arise that not only concern the precise definition of spatial arrangements, but also the identification of atom types and bond distances. Prominent examples include CO dehydrogenase, hydrogenase, acetylene hydratase and nitrogenase, all of which employ unique active sites that turned out not to be what they seemed upon first inspection.

Extended reaction scope of thiamine diphosphate dependent cyclohexane-1,2-dione hydrolase: from C-C bond cleavage to C-C bond ligation.

ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH) catalyzes the CC bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate. One of the two reactivities of CDH was selectively knocked down by mutation experiments. CDH-H28A is much less able to catalyze the CC bond formation, while the ability for CC bond cleavage is still intact. The double variant CDH-H28A/N484A shows the opposite behavior and catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol. Several acyloins of tertiary alcohols are formed with 54-94 % enantiomeric excess. In addition to pyruvate, methyl pyruvate and butane-2,3-dione are alternative donor substrates for CC bond formation. Thus, the very rare aldehyde-ketone cross-benzoin reaction has been solved by design of an enzyme variant.

Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins.

Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,ß-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

Catalytic scope of the thiamine-dependent multifunctional enzyme cyclohexane-1,2-dione hydrolase.

The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP-dependent enzymes. On a semipreparative scale, sterically demanding 4-(tert-butyl)benzaldehyde and 2-naphthaldehyde were transformed into the corresponding 2-hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.

The tricky task of nitrate/nitrite antiport.

Subtle differences: Two recent crystal structures have provided the first insight into nitrate/nitrite exchangers (example shown with bound nitrite), which are crucial to bacterial metabolism. A direct comparison of the structures reveals how the proteins can distinguish between their highly similar substrates and translate this into a conformational change to translocate ions across the membrane.

Analysis of the magnetic properties of nitrogenase FeMo cofactor by single-crystal EPR spectroscopy.

The catalytic center of nitrogenase, the [Mo:7Fe:9S:C]:homocitrate FeMo cofactor, is a S=3/2 system with a rhombic magnetic g tensor. Single-crystal EPR spectroscopy in combination with X-ray diffraction were used to determine the relative orientation of the g tensor with respect to the cluster structure. The protein environment influences the electronic structure of the FeMo cofactor, dictating preferred orientations of possible functional relevance.

The sixteenth iron in the nitrogenase MoFe protein.

Another iron in the fire: X-ray anomalous diffraction studies on the nitrogenase MoFe protein show the presence of a mononuclear iron site, designated as Fe16, which was previously identified as either Ca(2+) or Mg(2+). The position of the absorption edge indicates that this site is in the oxidation state +2. The high sequence conservation of the residues coordinated to Fe16 emphasizes the potential importance of the site in nitrogenase.

The formate/nitrite transporter family of anion channels.

The formate/nitrite transporter (FNT) family of integral membrane proteins comprises pentameric channels for monovalent anions that exhibit a broad specificity for small anions such as chloride, the physiological cargo molecules formate, nitrite, and hydrosulfide, and also larger organic acids. Three-dimensional structures are available for the three known subtypes, FocA, NirC, and HSC, which reveal remarkable evolutionary optimizations for the respective physiological context of the channels. FNT channels share a conserved translocation pathway in each protomer, with a central hydrophobic cavity that is separated from both sides of the membrane by a narrow constriction. A single protonable residue, a histidine, plays a key role by transiently protonating the transported anion to allow an uncharged species to pass the hydrophobic barrier. Further selectivity is reached through variations in the electrostatic surface potential of the proteins, priming the formate channel FocA for anion export, whereas NirC and HSC should work bidirectionally. Electrophysiological studies have shown that a broad variety of monovalent anions can be transported, and in the case of FocA, these match exactly the products of mixed-acid fermentation, the predominant metabolic pathway for most enterobacterial species.

α-Hydroxy-ß-keto acid rearrangement-decarboxylation: impact on thiamine diphosphate-dependent enzymatic transformations.

The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-ß-keto acid rather than a donor-acceptor substrate role variation. Further experiments with other ThDP-dependent enzymes, YerE, SucA, and CDH, verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations.