CHEMICAL MODIFICATION OF TYR34 AND TYR129 IN RABBIT LIVER MICROSOMAL CYTOCHROME B(5) AFFECTS INTERACTION WITH CYTOCHROME-P-450 2B4

Rabbit liver microsomal cytochrome b(5) was allowed to react with tetr anitromethane. Up to three tyrosine residues in each cytochrome b(5) m olecule were found to be accessible to the nitrating agent. Co-modific ation of tryptophan and histidine residues could be disregarded. CD-sp ectral measurements disproved gross changes in cytochrome b(5) structu re as a consequence of derivatization. Introduction of 1.6 nitro group s/polypeptide chain resulted in a fivefold increase in binding affinit y for cytochrome P-450 2B4 (P-450 2B4), whereas spectral interaction w ith cytochrome c remained unaffected. Furthermore, the capacity of nit rated cytochrome b(5) to shift the spin equilibrium to the high-spin c onformer of P-4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH-dependent electr on flow to ferric P-450 2B4. Changes in the redox potential of cytochr ome b(5) could be discounted as being responsible for this effect. The overall oxidative turnover of 4-nitroanisole did not respond to cytoc hrome b(5) modification. MS analysis and sequencing of peptide fragmen ts produced by tryptic digestion of modified cytochrome b(5) permitted the detection of three nitrated tyrosine residues located at position s 11, 34 and 129. Derivatization of cytochrome b(5) in the presence of a protective amount of P-450 2B4 provided evidence of the involvement of Tyr34 and Tyr129 in complexation of the two hemoproteins. It is pr oposed that Tyr129 might control docking of cytochrome b(5) to P-450 2 B4, whereas Tyr34 could be of functional importance in electron transf er.