The overall goal of this grant application is to investigate the structure and the function of the subunits of the Paracoccus NADH dehydrogenase complex. During the current granting period (2 years), we have identified the NADH-binding subunit of this enzyme complex utilizing direct photoaffinity labeling in the presence of [32 P]NADH. The labeled polypeptide was isolated by SDS gel electrophoresis and was shown to crossreact with antiserum to the NADH-binding subunit of the bovine NADH-ubiquinone oxidoreductase. In addition, we have cloned the structural gene of the NADH-binding subunit of the Paracoccus NADH dehydrogenase complex. The NADH-binding subunit has 431 amino acid residues and a calculated molecular weight of 47,191. The encoded protein contains a putative NAD(H)-binding and an iron-sulfur cluster-binding consensus sequence. Furthermore, when partial DNA sequencing of the nearby regions was performed, sequences homologous to the 24 kDa, the 49 kDa and the 75 kDa polypeptides of bovine complex I were found, suggesting that the structural genes of the Paracoccus NADH dehydrogenase complex construct at least one operon. Studies planned for this grant period are as follows: (i) Determination of the DNA sequence of the operon for the Paracoccus NADH dehydrogenase complex. When an unidentified open reading frame(s) is present in this operon, the presence of possible polypeptide(s) will be assessed using antiserum to an oligopeptide based on the deduced primary structure of the possible protein. (ii) Identification of the DCCD-binding and the rotenone-binding subunit(s) of the Paracoccus NADH dehydrogenase complex. (iii) Construction and characterization of NADH-binding subunit gene deletion mutants for the Paracoccus NADH dehydrogenase complex using gene replacement techniques.
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