Open AccessChemical Modification of Tyr34 and Tyr129 in Rabbit Liver Microsomal Cytochrome b 5 Affects Interaction with Cytochrome P ‐450 2B4
Author(s) -
Hlavica Peter,
Kellermann Josef,
Golly Ines,
Lehnerer Michael
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.01039.x
Subject(s) - cytochrome b5 , chemistry , cytochrome , hemeprotein , cytochrome c , cytochrome b , stereochemistry , heme , biochemistry , enzyme , mitochondrion , mitochondrial dna , gene
Rabbit liver microsomal cytochrome b 5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b 5 molecule were found to be accessible to the nitrating agent. Co‐modification of tryptophan and histidine residues could be disregarded. CD‐spectral measurements disproved gross changes in cytochrome b 5 structure as a consequence of derivatization. Introduction of 1.6 nitro groups/polypeptide chain resulted in a fivefold increase in binding affinity for cytochrome P ‐450 2B4 ( P ‐450 2B4), whereas spectral interaction with cytochrome c remained unaffected. Furthermore, the capacity of nitrated cytochrome b 5 to shift the spin equilibrium to the high‐spin conformer of P ‐4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH‐dependent electron flow to ferric P ‐450 2B4. Changes in the redox potential of cytochrome b 5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4‐nitroanisole did not respond to cytochrome b 5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b 5 permitted the detection of three nitrated tyrosine residues located at positions 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 proposed that Tyr129 might control docking of cytochrome b 5 to P ‐450 2B4, whereas Tyr34 could be of functional importance in electron transfer.