The NADP-reducing hydrogenase from Desulfovibrio fructosovorans: functional interaction between the C-terminal region of HndA and the N-terminal region of HndD subunits.
The hndABCD operon from Desulfovibrio fructosovorans encodes an uncommon heterotetrameric NADP-reducing iron hydrogenase. The presence of a [2Fe-2S] cluster likely located in the C-terminal region of the HndA subunit has already been revealed. We have cloned and expressed the truncated hndA gene in Escherichia coli to isolate the structural [2Fe-2S] module. Optical and EPR spectra are found identical to that of the native HndA subunit and the midpoint redox potential (-385 mV) is similar to that of the native protein (-395 mV). These results clearly demonstrate that the C-terminal region of HndA is a structurally independent [2Fe2S] ferredoxin-like domain. In the same way, the N-terminal domain of the HndD subunit was overproduced in E. coli and characterized. The presence of a [2Fe-2S] cluster was evidenced by optical spectroscopy. The midpoint redox potential (-380 mV) of this domain was found very close to that of the truncated HndA subunit but the EPR properties were significantly different. The various EPR properties allowed us to observe an electron exchange between the two [2Fe-2S] ferredoxin-like domains of the HndA and HndD subunits. Moreover, domain-domain interactions, observed by far-western experiments, indicate that these subunits are direct partners in the native complex.
Dermoun
Zorah
Z
Laboratoire de Bioénergétique et Ingénierie des Protéines, Centre National de la Recherche Scientifique, IBSM, 31 Chemin Joseph Aiguier, 13402 cedex 20, Marseille, France. dermoun@ibsm.cnrs-mrs.fr
De Luca
Gilles
G
Asso
Marcel
M
Bertrand
Patrick
P
Guerlesquin
Françoise
F
Guigliarelli
Bruno
B
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003901:Desulfovibrio / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D001426:Bacterial Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D010088:Oxidoreductases; C093595:NADP-reducing hydrogenase, Desulfovibrio fructosovorans