Azide binding and active site dynamics of position-82 variants of ferricytochrome c

Abstract

Azide binding to wild-type and five position-82 variants of yeast iso-l-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) ≈ Phe82Ile (64 M−1) < Phe82Leu (96 M−1) - Phe82Gly (105 M−1), pH 6, 25°C, μ = 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 60−1 (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.