The hydrogen binding site(s) in nickel hydrogenases (original) (raw)
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Nickel-Iron-Selenium Hydrogenases - An Overview
European Journal of Inorganic Chemistry, 2011
NiFeSe] hydrogenases are a subgroup of the large family of [NiFe] hydrogenases in which a selenocysteine ligand coordinates the Ni atom at the active site. As observed for other selenoproteins, the [NiFeSe] hydrogenases display much higher catalytic activities than their Cys-containing homologues. Here, we review the biochemical, catalytic, spectroscopic and structural properties of known [NiFeSe] hydrogenases, namely from the Hys (group 1 [NiFeSe] hydrogenase), Fru (F 420 -reducing [NiFeSe] hydrogenases) and Vhu families (F 420 -non-reducing [NiFeSe] hydrogenases). A survey of new [NiFeSe] hydrogenases present in the databases showed that all enzymes belong to either group 1 periplasmic uptake hydrogenases (Hys) or to group 3 cytoplasmic hydrogenases (Fru and Vhu) and are present in either sulfate-re-Nickel-Iron-Selenium Hydrogenases pool. They are characterized by the presence of a signal peptide in the small subunit, involved in translocation of the two mature subunits to the periplasm. Most group 1 Hases are associated with a third subunit, cytochrome b, responsible for membrane anchoring and quinone reduction, with the exception of periplasmic Hases of Desul-Carla S. A. Baltazar received her Bachelors and Masters degrees in Biochemistry from the Faculty of Sciences at the University of Lisbon, Portugal in 2006 and 2008, respectively. She then became a Research Student in the Protein Modelling Laboratory at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, under the supervision of Dr. Cláudio Soares. Currently, she is studying H 2 diffusion and proton pathways in [NiFeSe] hydrogenases using computational methodologies, such as Molecular Dynamics and Continuum Electrostatics. Marta Marques graduated from the Instituto Superior Técnico in 2007 with an M.Sci. degree in Biological Engineering. She started working in 2008 as a research student in the Instituto de Tecnologia Química e Biológica at the Universidade Nova de Lisboa. In 2010, she obtained a Ph.D. research grant from the Fundação para a Ciência e Tecnologia (FCT, MCES, Portugal), under the supervision of Drs. Inês Pereira and Pedro Matias. Her research work is focused on the functional, structural and spectroscopic characterization of the [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough, which combines the use of biochemistry, spectroscopy, X-ray crystallography, molecular biology and electrochemistry, in order to understand the molecular basis for the remarkable catalytic properties of this hydrogenase. Dr. Cláudio M. Soares received a Licenciado degree in
Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1986
A preparation of hydrogenase from WolineUa (formerly Vibrio) succinogenes enriched in 33S to at least 70% has been studied by EPR spectroscopy. The sulphur isotope, which has a nuclear spin of 3/2, clearly broadened the spectrum of the Fe-S cluster. Resolved hyperfine splitting was observed in one of the lines of the EPR spectrum of Ni(III). It was concluded that this was due to interaction with one 33S nucleus. Also the appearance of the high-field line of the EPR signal of Ni(1), either before or after photodissociation of the nickel-hydrogen bond, indicated hyperfine interaction with one 33S nucleus. No indication has been observed for nitrogen hyperfine interaction in any of the EPR spectra in this study. The data provide independent proof of earlier conclusions from EXAFS studies by two other groups (
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1996
Upon reduction under hydrogen-argon atmosphere, the nickel-hydrogenases generally show a characteristic rhombic EPR spectrum which is known as Ni-C. Illumination of this state at temperatures below 60 K has previously been shown to cause the disappearance of the Ni-C signal and the simultaneous appearance of two overlapping signals, here referred to as Ni-L1 and Ni-L2, which revert to the Ni-C state at higher temperatures. These phenomena have been compared in three nickel-containing hydrogenases, the [NiFe]-hydrogenases from Desulfot'ibrio gigas and Desulfovibrio fructosovorans, and the selenium-containing soluble [NiFeSe]-hydrogenase from Desulfomicrobium baculatum. Significant differences were observed between these enzymes. (1) The Ni-C, Ni-L1 and Ni-L2 EPR spectra were almost identical for D. gigas and D. fructosovorans hydrogenases, but the rates of photoconversion of Ni-C to Ni-L1 were different, being about 5 times slower for D. fructosovorans than for D. gigas in H20. The kinetic isotope effect in 2H20/HzO was a factor of thirty in D. gigas, but only two in D. fructosovorans. Dm. baculatum hydrogenase showed almost no kinetic isotope effect on the Ni-C to Ni-L1 conversion, but an effect on the conversion of Ni-L1 to Ni-L2. The kinetic isotope effects indicate that the processes involve the movement of a hydrogen nucleus. (3) The Ni-L1 species converted to into Ni-L2 in the dark at a rate that was virtually temperature-independent below 30 K, indicative of a proton tunnelling process. (4) The conversion of Ni-L1 to Ni-L2 was partly reversed by light in Dm. baculatum hydrogenase, but not in the [NiFe]-hydrogenases. (5) Prolonged illumination of the three enzymes induced the appearance of a third light-induced signal, Ni-L3. The new signal was rhombic, with features at g = 2.41, 2.16 (the third component being unresolved) in the [NiFe]-hydrogenases and g = 2.48, 2.16, 2.03 for the [NiFeSe]-enzyme. (6) Splittings caused by by spin-spin interactions with [4Fe-4S] clusters were detected for all the illuminated signals, Ni-LI, Ni-L2 and Ni-L3. These were quantitatively different for the three enzymes. (7) Broadening of the Ni-C signals in H20 compared with 2H20 was observed in the gj and g2 components of D. gigas and D. fructosovorans hydrogenases, but not for Din. baculatum. This broadening effect was not seen with any of the Ni-L species. These comparative effects are discussed in terms of subtle differences in the structure and protein environment of the nickel site, and access to exchangeable hydrons.
FEBS Letters, 2005
The large subunit HoxC of the H 2 -sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.
FEBS Letters, 2000
The steps in the maturation of the precursor of the large subunit (pre-HycE) of hydrogenase 3 from Escherichia coli taking place after incorporation of both iron and nickel were investigated. Pre-HycE could be matured and processed in the absence of the small subunit but association with the cytoplasmic membrane required heterodimer formation between the two subunits. Pre-HycE formed a complex with the chaperone-like protein HypC in the absence of the small subunit and, in this complex, also incorporated nickel. For the C-terminal processing, HypC had to leave the complex since only a HypC-free, nickel-containing form of pre-HycE was a substrate for the maturation endopeptidase.
Journal of The American Chemical Society, 2000
L-edge X-ray absorption spectroscopy has been used to study, under a variety of conditions, the electronic structure of Ni in the Ni-Fe hydrogenases from DesulfoVibrio gigas, DesulfoVibrio baculatus, and Pyrococcus furiosus. The status of the enzyme films used for these measurements was monitored by FT-IR spectroscopy. The L-edge spectra were interpreted by ligand field multiplet simulations and by comparison with data for Ni model complexes. The spectrum for Ni in D. gigas enzyme "form A" is consistent with a covalent Ni(III) species. In contrast, all of the reduced enzyme samples exhibited high spin Ni(II) spectra. The significance of the Ni(II) spin state for the structure of the hydrogenase active site is discussed. Frey, M.; Garcin, E.; Hatchikian, C.; Montet, Y.; Piras, C.; Vernede, X.; Volbeda, A.
Biochemistry, 2000
An X-ray absorption spectroscopic study of structural changes occurring at the Ni site of Chromatium vinosum hydrogenase during reductive activation, CO binding, and photolysis is presented. Structural details of the Ni sites for the ready silent intermediate state, SI(r), and the carbon monoxide complex, SI-CO, are presented for the first time in any hydrogenase. Analysis of nickel K-edge energy shifts in redox-related samples reveals that reductive activation is accompanied by an oscillation in the electron density of the Ni site involving formally Ni(III) and Ni(II), where all the EPR-active states (forms A, B, and C) are formally Ni(III), and the EPR-silent states are formally Ni(II). Analysis of XANES shows that the Ni site undergoes changes in the coordination number and geometry that are consistent with five-coordinate Ni sites in forms A, B, and SI(u); distorted four-coordinate sites in SI(r) and R; and a six-coordinate Ni site in form C. EXAFS analysis reveals that the loss of a short Ni-O bond accounts for the change in coordination number from five to four that accompanies formation of SI(r). A shortening of the Ni-Fe distance from 2.85(5) A in form B to 2.60(5) A also occurs at the SI level and is thus associated with the loss of the bridging O-donor ligand in the active site. Multiple-scattering analysis of the EXAFS data for the SI-CO complex reveals the presence of Ni-CO ligation, where the CO is bound in a linear fashion appropriate for a terminal ligand. The putative role of form C in binding H(2) or H(-) was examined by comparing the XAS data from form C with that of its photoproduct, form L. The data rule out the suggestion that the increase in charge density on the NiFe active site that accompanies the photoprocess results in a two-electron reduction of the Ni site [Ni(III) --> Ni(I)] [Happe, R. P., Roseboom, W., and Albracht, S. P. J. (1999) Eur. J. Biochem. 259, 602-608]; only subtle structural differences between the Ni sites were observed.