AZIDE BINDING AND ACTIVE SITE DYNAMICS OF POSITION-82 VARIANTS OF FERRICYTOCHROME C

摘要

Azide binding to wild-type and five position-82 variants of yeast iso-1-ferricytochrome c has been studied by spectrophotometric titrations and stopped-flow kinetics to determine the extent to which mutations that affect the active-site structure of this protein modulate interaction with heme binding ligands. The variant proteins exhibit greater affinity for azide than does the wild-type protein [wild-type (16.7 M(-1)) < Phe82Tyr (31 M(-1)) < Phe82Ser (56 M(-1)) approximate to Phe82Ile (64 M(-1)) < Phe82Leu (96 M(-1)) approximate to Phe82Gly (105 M(-1)), pH 6, 25 degrees C, mu = 1.0 M]. The affinity of cytochrome c for azide correlates with the relative stability of the variant to formation of the alkaline conformational state. The kinetics of azide binding to ferricytochrome c are consistent with a two-step, reversible mechanism that exhibits rate saturation with increasing azide concentration. The limiting forward rate constants range from 60 s(-1) (wild-type cytochrome) to 360 s(-1) (Phe82Ser variant). The results of these thermodynamic and kinetic studies are interpreted in terms of the known or likely structures of the variants. [References: 40]