Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli.
Based on the DNA sequence of its structural genes, clustered in the hnd operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovorans is thought to be a heterotetrameric complex in which HndA and HndC constitute the NADP-reducing unit and HndD constitutes the hydrogenase unit, respectively. The weak representativity of the enzyme among cell proteins has prevented its purification. This paper discusses the purification and characterization of the HndA subunit of this unique tetrameric iron hydrogenase overproduced in Escherichia coli. The purified subunit contains 1.7 mol of non-heme iron and 1.7 mol of acid-labile sulfide/mol. EPR analysis of the reduced form of HndA indicates that it contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with values of gx, gy, and gz equal to 1.915, 1.950, and 2. 000, respectively, and a midpoint redox potential of -395 mV. The UV-visible and EPR spectra of the [2Fe-2S] cluster indicate that HndA belongs to the [2Fe-2S] family typified by the Clostridium pasteurianum [2Fe-2S] ferredoxin. The C-terminal sequence of HndA shows 27% identity with the C-terminal sequence of the 25-kDa subunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from Bos taurus complex I, and 32% identity with the entire sequence of C. pasteurianum [2Fe-2S] ferredoxin. The four cysteine residues involved in HndA cluster binding have been tentatively identified on the basis of sequence identity considerations. Evidence of a HndA organization based on two independent structural domains is discussed.
De Luca
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Laboratoire de Bioenergetique et Ingenierie des Proteines, Centre National de la Recherche Scientifique, IFR C1, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. deluca@ibsm.cnrs-mrs.fr
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Belaich
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Dermoun
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003013:Clostridium / Q000201:enzymology / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D003901:Desulfovibrio / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D010231:Paracoccus denitrificans / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C093595:NADP-reducing hydrogenase, Desulfovibrio fructosovorans