J. Appl. Cryst.の発刊に際して

Sustainable hydrogen production is an essential prerequisite of a future hydrogen economy. Water electrolysis driven by renewable resource-derived electricity and direct solar-to-hydrogen conversion based on photochemical and photoelectrochemical water splitting are promising pathways for sustainable hydrogen production. All these techniques require, among many things, highly active noble metal-free hydrogen evolution catalysts to make the water splitting process more energy-efficient and economical. In this review, we highlight the recent research efforts toward the synthesis of noble metal-free electrocatalysts, especially at the nanoscale, and their catalytic properties for the hydrogen evolution reaction (HER). We review several important kinds of heterogeneous non-precious metal electrocatalysts, including metal sulfides, metal selenides, metal carbides, metal nitrides, metal phosphides, and heteroatom-doped nanocarbons. In the discussion, emphasis is given to the synthetic methods of these HER electrocatalysts, the strategies of performance improvement, and the structure/composition-catalytic activity relationship. We also summarize some important examples showing that non-Pt HER electrocatalysts could serve as efficient cocatalysts for promoting direct solar-to-hydrogen conversion in both photochemical and photoelectrochemical water splitting systems, when combined with suitable semiconductor photocatalysts.

Noble metal-free hydrogen evolution catalysts for water splitting

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

We are just beginning to understand the metabolism of heavy metals and to use their metabolic functions in biotechnology, although heavy metals comprise the major part of the elements in the periodic table. Because they can form complex compounds, some heavy metal ions are essential trace elements, but, essential or not, most heavy metals are toxic at higher concentrations. This review describes the workings of known metal-resistance systems in microorganisms. After an account of the basic principles of homoeostasis for all heavy-metal ions, the transport of the 17 most important (heavy metal) elements is compared.

Microbial heavy-metal resistance

Heme-Containing Oxygenases

Structure−Function Relationships of Alternative Nitrogenases

The requirement for molybdenum in biological dinitrogen fixation, first reported by Bortels1, is due to its involvement at or near the site of reduction of N2 in conventional nitrogenase. To date, all nitrogenases which have been purified to homogeneity consist of an iron protein (component 2) and a molybdoprotein (component 1)2. Azotobacter vinelandii, an obligately aerobic diazotrophic bacterium, has two systems for nitrogen fixation: a conventional nitrogenase involving molybdenum and an alternative system which functions under conditions of Mo deficiency and does not require the structural genes for conventional nitrogenase3–6. The properties of the nitrogenase in extracts of comparable deletion strains of A. vinelandii are consistent with a two-component system6,7 in which the component 1-containing fraction has no detectable Mo (ref. 6). Recently, an alternative nitrogen fixation system has been demonstrated in Azotobacter chroococcum strain MCD1155, in which the structural genes for conventional nitrogenase are deleted8. We demonstrate here that nitrogen fixation by this strain depends on vanadium and we show that its purified nitrogenase is a binary system in which the conventional molybdoprotein is replaced by a vanadoprotein.

The alternative nitrogenase of Azotobacter chroococcum is a vanadium enzyme

Current status of structure function relationships of vanadium nitrogenase

1. Nitrogenase from the facultative anaerobe Klebsiella pneumoniae was resolved into two protein components resembling those obtained from other nitrogen-fixing bacteria. 2. Both proteins were purified to homogeneity as shown by the criteria of disc electrophoresis and ultracentrifugal analysis. 3. The larger component had a mol.wt. of 218000 and contained one Mo atom, 17Fe atoms and 17 acid-labile sulphide groups/mol; it contained two types of subunit, present in equal amounts, of mol.wts. 50000 and 60000. All the common amino acids were present, with a predominance of acidic residues. The apparent partial specific volume was 0.73; ultracentrifugal analysis gave s020,w=11.0S and D020,w=4.94×10−7cm2/s. The specific activities (nmol of product formed/min per mg of protein) when assayed with the second nitrogenase component were 1500 for H2 evolution, 380 for N2 reduction, 1200 for acetylene reduction and 5400 for ATP hydrolysis. The reduced protein showed electron-paramagnetic-resonance signals at g=4.3, 3.7 and 2.015; the Mossbauer spectrum of the reduced protein consisted of at least three doublets. The u.v. spectra of the oxidized and reduced proteins were identical. On oxidation the absorbance increased generally throughout the visible region and a shoulder at 430nm appeared. The circular-dichroism spectra of both the oxidized and reduced proteins were the same, consisting mainly of a negative trough at 220nm. 4. The smaller component had mol.wt. 66800 and contained four Fe atoms and four acid-labile sulphide groups in a molecule comprising two subunits each of mol.wt. 34600. All common amino acids except tryptophan were present, with a predominance of acidic residues. The apparent partial specific volume calculated from the amino acid analysis was 0.732, which was significantly higher than that obtained from density measurements (0.69); ultracentrifugal analysis gave s020,w=4.8S and D020,w=5.55×10−7cm2/s. The specific activities (nmol of product formed/min per mg of protein) were 1050 for H2 evolution, 275 for N2 reduction, 980 for acetylene reduction and 4350 for ATP hydrolysis. The protein was not cold-labile. The reduced protein showed electron-paramagnetic-resonance signals in the g=1.94 region. The Mossbauer spectrum of the reduced protein consisted of a doublet at 77°K. The u.v. spectra of reduced and O2-inactivated proteins were identical, and inactivation by O2 generally increased the absorbance in the visible region and resulted in a shoulder at 460nm. The circular-dichroism spectra exhibited a negative trough at 220nm and inactivation by O2 decreased the depth of the trough. 5. The reduction of N2 and acetylene, and H2 evolution, were maximal at a 1:1 molar ratio of the Fe-containing protein to the Mo–Fe-containing protein; excess of the Mo–Fe-containing protein was inhibitory. All reductions were accompanied by H2 evolution. The combined proteins had no ATP-independent hydrogenase activity.

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Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins.

Gene fusions in which the lac genes are under the control of each promoter in the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster have been constructed. These fusions have been used to examine positive control of the cluster and the response of individual genes to repression by ammonia and oxygen. De-repression of nif transcriptional units is coordinate and molybdate is required for maximal expression of the structural gene operon, which is autogenously regulated.

Robert R. Eady

Papers

Structure−Function Relationships of Alternative Nitrogenases

The alternative nitrogenase of Azotobacter chroococcum is a vanadium enzyme

Current status of structure function relationships of vanadium nitrogenase

Nitrogenase of Klebsiella pneumoniae. Purification and properties of the component proteins.

Analysis of regulation of Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions