The multiple nicotinamide nucleotide‐binding subunits of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I)

Direct photoaffinity labeling of purified bovine heart NADH:ubiquinone oxidoreductase (complex I) with 32P‐labeled NAD(H), NADP(H) and ADP has shown that five polypeptides become labeled, with molecular masses of 51, 42, 39, 30, and 18–20 kDa. The 51 and the 30‐kDa polypeptides were labeled with either [32P]NAD(H), [32P]NADP(H) or [β‐32P]ADP. The 42‐kDa polypeptide was labeled with [32P]NAD(H) and to a small extent with [β‐32P]ADP. It was not labeled with [32P]NADP(H). The 39‐kDa polypeptide was labeled with [32P]NADPH and to a small extent with [β‐32P]ADP. Our previous studies had shown that this subunit also binds NADP, but not NAD(H) [Yamaguchi, M., Belogrudov, G.I. & Hatefi, Y. (1998) J. Biol. Chem.273, 8094–8098]. The 18–20‐kDa polypeptide was labeled only with [32P]NADPH. Among these polypeptides, the 51‐kDa subunit is known to contain FMN and a [4Fe–4S] cluster, and is the NAD(P)H‐binding subunit of the primary dehydrogenase domain of complex I. The possible roles of the other nucleotide‐binding subunits of complex I have been discussed.