Purification and Properties of a Cytochrome b5-Like Hemeprotein from Mitochondrial Outer Membranes of Rat Liver

Abstract

Upon solubilization with sodium cholate, the cytochrome b⁵-like hemeprotein associated with the outer membranes of rat liver mitochondria (abbreviated here as OM-cytochrome b⁵) could be preferentially released from sonicated mitochondrial outer membrane preparations. The OM-cytochrome b⁵ thus solubilized was highly purified by DEAE-cellulose, DEAE-Sepharose CL-6B, hydroxylapatite column chromatography in the presence of 0.5% sodium cholate and the isoelectric focusing method. Sepharose 6B gel chromatography in the presence of 6 M guanidine hydrochloride and Sephadex G-75 gel chromatography in the presence of 0.5% sodium dodecyl sulfate (SDS) indicated a monomeric molecular weight of about 16,000 for the detergent-solubilized OM-cytochrome b⁵. However, in aqueous solutions, this cytochrome occurs as a stable polymeric species since its molecular weight has been found to be approximately 60,000 even in 0.5% sodium cholate. The absorption spectrum of reduced OM-cytochrome b⁵ in the visible region closely resembled that of microsomal cytochrome b⁵, however, the OM-cytochrome b⁵ showed a broad, symmetrical α-band with a maximum around 557 nm. A high level of NADH-cytochrome c reductive activity was detected by the addition of both the purified L-kynurenine 3-hydroxylase [EC 1.14.13.9] and OM-cytochrome b⁵ preparation to a sonicated phospholipids liposome bilayer, although only the purified monooxygenase or OM-cytochrome b⁵ could not catalyze the reduction of cytochrome c by NADH. The rate of NADH-cytochrome c reduction catalyzed by the monooxygenase and this hemeprotein in the presence of PC liposomes was affected by high concentrations of rotenone or dicumarol.