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Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli.
Biochemistry. 1998 Feb 24; 37(8):2660-5.B

Abstract

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.

Authors+Show Affiliations

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.frNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9485416

Citation

De Luca, G, et al. "Purification and Characterization of the HndA Subunit of NADP-reducing Hydrogenase From Desulfovibrio Fructosovorans Overproduced in Escherichia Coli." Biochemistry, vol. 37, no. 8, 1998, pp. 2660-5.
De Luca G, Asso M, Belaich JP, et al. Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli. Biochemistry. 1998;37(8):2660-5.
De Luca, G., Asso, M., Belaich, J. P., & Dermoun, Z. (1998). Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli. Biochemistry, 37(8), 2660-5.
De Luca G, et al. Purification and Characterization of the HndA Subunit of NADP-reducing Hydrogenase From Desulfovibrio Fructosovorans Overproduced in Escherichia Coli. Biochemistry. 1998 Feb 24;37(8):2660-5. PubMed PMID: 9485416.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification and characterization of the HndA subunit of NADP-reducing hydrogenase from Desulfovibrio fructosovorans overproduced in Escherichia coli. AU - De Luca,G, AU - Asso,M, AU - Belaich,J P, AU - Dermoun,Z, PY - 1998/3/28/pubmed PY - 1998/3/28/medline PY - 1998/3/28/entrez SP - 2660 EP - 5 JF - Biochemistry JO - Biochemistry VL - 37 IS - 8 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9485416/Purification_and_characterization_of_the_HndA_subunit_of_NADP_reducing_hydrogenase_from_Desulfovibrio_fructosovorans_overproduced_in_Escherichia_coli_ L2 - https://doi.org/10.1021/bi972474p DB - PRIME DP - Unbound Medicine ER -