Inverse relationship between cytochrome P-450 phosphorylation and complexation with cytochrome b5.

Cytochrome P-450 LM2 purified from rabbit liver microsomes has been shown to be a substrate for cAMP-dependent protein kinase. Cytochrome b5, in contrast, was a very poor substrate for cAMP-dependent protein kinase, although it stimulated the activity of the kinase toward histone. When purified rabbit cytochrome b5 was mixed with purified LM2, phosphorylation of LM2 by cAMP-dependent protein kinase was inhibited approximately 80-90%. Recently, a functional covalent complex of cytochrome b5 and LM2 was prepared and purified to homogeneity (P.P. Tamburini and J.B. Schenkman (1987) Proc. Natl. Acad. Sci. USA 84, 11-15). When present as a covalent complex with cytochrome b5, the phosphorylation of LM2 in the complex by cAMP-dependent protein kinase was also inhibited about 80-90% relative to an equivalent amount of LM2 alone. On the other hand, when the LM2 was phosphorylated prior to interaction with cytochrome b5, the ability of the latter to perturb the spin equilibrium of LM2 and oxidation of p-nitroanisole by the LM2 was diminished to an extent comparable to the degree of phosphorylation. The results suggest either that the phosphorylation site on LM2 may be within the cytochrome b5 binding site or that phosphorylation and cytochrome b5 cause mutually exclusive conformational changes in LM2. In addition, eight different forms of cytochrome P-450 from the rat (RLM2, RLM3, fRLM4, RLM5, RLM5a, RLM5b, RLM6, and PBRLM5) were examined as potential substrates for cAMP-dependent protein kinase under the same conditions. Maximal phosphorylation of about 20 mol% was obtained with LM2, and about half as much with PBRLM5. The low extent of phosphorylation of LM2 was not due to the prior presence of phosphate on the enzyme since LM2, as isolated, contains less than 0.1 mol phosphate/mol of enzyme. The other forms of cytochrome P-450 tested showed little or no phosphorylation in vitro despite the presence of a cAMP-dependent protein kinase phosphorylation sequence on at least two of them.