Similarities in the Architecture of the Active Sites of Ni-Hydrogenases and Fe-Hydrogenases Detected by Means of Infrared Spectroscopy (original) (raw)
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European Journal of Biochemistry, 1990
The soluble (cytoplasmic plus periplasmic) Ni/Fe-S/Se-containing hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from cells grown in an 57Fe-enriched medium, and its iron-sulfur centers were extensively characterized by Mossbauer and EPR spectroscopies. The data analysis excludes the presence of a [3Fe-4S] center, either in the native (as isolated) or in the hydrogen-reduced states. In the native state, the non-heme iron atoms are arranged as two diamagnetic [4Fe-4SI2+ centers. Upon reduction, these two centers exhibit distinct and unusual Mossbauer spectroscopic parameters. The centers were found to have similar mid-point potentials ( x -31 5 mV) as determined by oxidation-reduction titrations followed by EPR.
Biochemical and Biophysical Research Communications, 1997
rium, with different cellular localizations, is yet poorly The [NiFe] hydrogenase from Desulfovibrio vulgaris understood. It may reflect a mechanism for metabolic Hildenborough was isolated from the cytoplasmic regulation, which might depend on the growth condimembranes and characterized by EPR spectroscopy. tions. For example, the requirement of two hydro-It has a total molecular mass of 98.7 kDa (subunits of genases for the reduction of sulfate to sulfite may be 66.4 and 32.3 kDa), and contains 1 nickel and 12 Fe fundamental in situations where sulfate is limiting atoms per heterodimer. The catalytic activities for hy-(4,5). The presence of multiple hydrogenases is a key drogen consumption and production were determined feature of the hydrogen-cycling hypothesis, a proposed to be 174 and 89 mmol H 2 rmin 01 rmg 01 , respectively. As bioenergetic mechanism in Desulfovibrio in which moisolated, under aerobic conditions, this hydrogenase lecular hydrogen is involved in the generation of a proexhibits EPR signals characteristic of the nickel centon motive force (4). ters in [NiFe] hydrogenases (Ni-A signal at g x,y,z Å2.32, D. vulgaris Hildenborough (DvH) contains the genes 2.23 and Ç2.0 and Ni-B signal at g x,y,z Å2.33, 2.16 and for the three different hydrogenases (3). The periplas-Ç2.0) as well as an intense quasi-isotropic signal cenmic [Fe] hydrogenase was the first to be purified and tered at gÅ2.02 due to the oxidized [3Fe-4S] center. The has been extensively characterized (1,6). The presence redox profile under hydrogen atmosphere is remarkably similar to that of other [NiFe] hydrogenases. The of [NiFe] and [NiFeSe]
The hydrogenases of DesuEfovibrio vulgaris and Megasphaera elsdenii are compared with respect to some of their physical properties. In addition to Fe the only metal ions that are present in significant amounts are Ni and Cu. From cluster extrusion experiments it follows that the D. vulgaris enzyme contains three 4 Fe-4 S clusters, while M . elsdenii hydrogenase only releases part of its Fe-S clusters. The resting D . vulgarisenzyme shows only a small 3 Fe-xS type of EPR signal (maximum 5 %electronequivalent). This amount can beincreased to approximately25 %by treatment with ferricyanide, with a concomitant large decrease in activity. The M. elsdenii enzyme shows in its oxidized state a normal Hipip (high-potential iron-sulphur protein) type of EPR spectrum. After a reduction/oxidation cycle the D. vulgaris enzyme also shows a weak Hipip type of EPR spectrum. In the reduced state both enzymes show complex spectra. By integration of those spectra it is shown that 1.5 electron equivalents are present. The complex spectra do not arise from nuclear hyperfine interactions but are partially due to electron spin interactions. It is proposed that the spectrum of reduced D. vulgaris hydrogenase consists of a sum of three different ferredoxin-like spectra.
The redox properties of the iron-sulphur cluster in hydrogenase from Chromatium vinosum, strain D
Biochimie, 1986
The midpoint potentials of the changes in the electron spin resonance (ESR) spectra in the region of g = 2 in hydrogenase II from Chromatium vinosum were estimated by redox titrations. As the enzyme was progressively reduced, the g = 2.02 signal increased, while the satellite lines at g = 1.98 etc. decreased. At still lower potentials the signal at g = 2.02 decreased. The midpoint potentials of the two processes were estimated to be + 100 mY and -20 mY, respectively, at pH 8.5. The first potential showed significant pl-l-dependence. The titration data fitted to n = I curves with reasonable reversibility. The enzyme activity showed no significant changes in this potential range. The results are discussed in relation to the interaction of the iron-sulphur cluster with nickel.
Fe-only hydrogenases: structure, function and evolution
Journal of Inorganic Biochemistry, 2002
Hydrogenases are enzymes capable of catalyzing the oxidation of molecular hydrogen or its production from protons and electrons 1 2 according to the reversible reaction: H á2H 12e . Most of these enzymes fall into to major classes: NiFe and Fe-only hydrogenases.
Proceedings of the National Academy of Sciences, 1983
Two hydrogenases from the methanogenic bacterium Methanobacterium thermoautotrophicum strain AH have been purified and contain tightly bound nickel as well as the anticipated iron/sulfur atoms with a fixed ratio of 15-20 iron atoms per nickel. One hydrogenase reduces the 8-hydroxy-5-deazaflavin coenzyme factor 420 (F420), whereas the other has been purified as a methyl viologen-reducing hydrogenase. Both enzymes possess an EPR signal attributed to paramagnetic nickel as demonstrated by hyperfine coupling in 6tNi-containing hydrogenases. Comparison to model compounds suggests a nickel(I) oxidation state in the inactive forms of these aerobically purified enzymes. Loss of the nickel(I][) signal accompanies reductive activation but is not kinetically correlated with regain of high specific activity. On replacement of H2 by argon in the gas phase over reduced, active, F420-reducing enzyme, several EPR signals appear, including a signal at g = 2.004 that is probably enzyme-bound FADH semiquinone, two signals at g = 2.140 and 2.196 that reflect a new form ofparamagnetic nickel(IE), and also a signal at g = 2.036 that may be an iron signal. The F420-reducing hydrogenase in the second paramagnetic nickel form is either itself active or in facile equilibrium with active enzyme. The size ofthe signal atg = 2.036 may correlate with the degree of activation of the enzyme. In contrast to the hydrogenase of Clostridium pasteurianum [Erbes, D. L., Burris, R. H. & Orme-johnson, W. H. (1975) Proc NatL Acad Sci USA 72, 4795-4799], which appears to use only iron/sulfur prosthetic groups and which reacts with one-electron-transfer agents, this methanogen hydrogenase seems to utilize iron, nickel, and flavin redox sites and to reduce obligate one-electron (viologen) and two-electron (deazaflavin) oxidants. Methanogenic bacteria reduce CO2 to CH4 in an overall eightelectron reduction process that involves the cooxidation of four molecules of hydrogen gas, H2.