Chemical modification of Tyr34 and Tyr129 in rabbit liver microsomal cytochrome b5 affects interaction with cytochrome P-450 2B4.
Rabbit liver microsomal cytochrome b5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b5 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 b5 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 b5 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 b5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4-nitroanisole did not respond to cytochrome b5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b5 permitted the detection of three nitrated tyrosine residues located at positions 11, 34 and 129. Derivatization of cytochrome b5 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 b5 to P-450 2B4, whereas Tyr34 could be of functional importance in electron transfer.
Hlavica
P
P
Walther Straub-Institut für Pharmakologie und Toxikologie, Universität München, Germany.
Kellermann
J
J
Golly
I
I
Lehnerer
M
M
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D004579:Electron Transport; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009566:Nitrates / Q000737:chemistry; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000378:metabolism*
D009566:Nitrates; D014443:Tyrosine; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System