PROTEIN AND GENE STRUCTURE OF THE NADH-BINDING FRAGMENT OF RHODOBACTER-CAPSULATUS NADH-UBIQUINONE OXIDOREDUCTASE

Membranes of aerobically grown Rhodobacter capsulatus contain only one type of NADH:ubiquinone oxidoreductase which is homologous to the pro ton-translocating complex I. The K-m value of the enzyme for NADH was determined to be 8 mu M. After solubilization of the membranes with an alkylglucoside detergent, two fragments of complex I with molecular m asses of 110 kDa and 140 kDa were isolated by chromatographic steps in the presence of detergent. Both fragments contain at least two polype ptides with apparent molecular masses of 46 kDa and 42 kDa. FMN was id entified as cofactor in the preparations. Degenerative oligonucleotide primers were used to amplify a past of the sequence coding for the NA DH-binding subunit of complex I by PCR. With the PCR product as probe, a genomic fragment was cloned and sequenced containing the genes enco ding the two purified polypeptides and additional reading frames. The two genes are named nuoE and nuoF and are homologous to nqo2 and nqo1 of Paracoccus denitrificans. However, NuoE contains a C-terminal exten sion of 149 amino acids compared with Nqo2, NuoE and NuoF have molecul ar masses of 41259 Da and 47133 Da and contain the NADH-, FMN- and FeS -cluster-binding motifs.