Active Complex between Adrenodoxin Reductase and Adrenodoxin in the Cytochrome P-450scc Reduction Reaction1

In order to elucidate the mechanism of the electron transfer reaction of mitochondrial steroid hydroxylase, the reduction reaction of cytochrome P-450scc (P-450scc) catalyzed by covalently cross-linked complexes between adrenodoxin reductase (AR) and adrenodoxin (AD) was studied. The reduction rate with the covalent AR-AD complex was very slow (0.030 min−1 as the flavin turnover number) compared with the reduction catalyzed by AR and AD (4.6 min−1). When free AD was added to the reaction mixture containing the AR-AD complex, the rate increased about 30 times. The AD dimer [(AD)2], and a complex between AR and the AD dimer [AR-(AD)2] were then prepared. The Vmax for the P-450scc reduction activity of AR with (AD)2 was 50% of that of AR with AD. The Km value for the total concentration of AD in the P-450scc reduction reaction mixture containing AR and (AD)2 was found to be the same as that in the reaction mixture containing AR and AD. P-450scc reduction by AR-(AD)2 was about 5 times faster than that by AR-AD. The addition of free AD to the AR-(AD)2 complex enhanced the P-450scc reduction about 30 times. AR-AD and AR-(AD)2 were able to reduce external AD, cytochrome c, and acetylated cytochrome c. From these results, it is concluded that organized complexes consisting of AR and AD are more active in the presence of free AD than in its absence, in terms of the reductive activity toward P-450scc. It was confirmed that the properties of P-450scc reduction by covalent complexes differ from those of cytochrome c reduction: the addition of free AD stimulates the reduction of P-450scc whereas it does not stimulate the reduction of cytochrome c.