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 hn d operon, the NADP-reducing hydrogenase of Desulfovibrio fructosovoran s is thought to be a heterotetrameric complex in which HndA and HndC c onstitute 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 pur ification and characterization of the HndA subunit of this unique tetr americ iron hydrogenase overproduced in Escherichia call, 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 i t contains a single binuclear [2Fe-2S] cluster. This cluster exhibits a spectrum of rhombic symmetry with values of g(x), g(y), and g(z) equ al to 1.915, 1.950, and 2.000, respectively, and a midpoint redox pote ntial of -395 mV. The UV-visible and EPR spectra of the [2Fe-2S] clust er indicate that HndA belongs to the [2Fe-2S] family typified by the C lostridium pasteurianum [2Fe-2S] ferredoxin, The C-terminal sequence o f HndA shows 27% identity with the C-terminal sequence of the 25-kDa s ubunit of NADH: quinone oxidoreductase from Paracoccus denitrificans, 33% identity with the C-terminal sequence of the 24-kDa subunit from B os taurus complex I, and 32% identity with the entire sequence of C. p asteurianum [2Fe-2S] ferredoxin. The four cysteine residues involved i n HndA cluster binding have been tentatively identified on the basis o f sequence identity considerations. Evidence of a HndA organization ba sed on two independent structural domains is discussed.