Reaction of cythchrome c with one-electron redox reagents. I. Reduction of ferricytochrome c by the hydrated electron produced by pulse radiolysis

Pulse radiolysis-kinetic spectrometry has been used to investigate the reaction of hydrated electrons with ferricytochrome c in dilute aqueous solution at pH 6.5–7.0. Time resolutions from 2·10^?7 to 1 s were employed. Transient spectra from 320 to 580 nm were characterized with a wavelength resolution of ±0.5 nm. 1 In neutral salt-free solution, k(ferricytochrome c+e^?_aq)=(6.0±0.9)·10¹⁰ M^?1·s^?1 and k(ferricytochrome c+H)=(1.2±0.2)·10¹⁰ M^?1·s^?1. The reaction of ferricytochrome c with hydrated electrons is sensitive to ionic strength; in 0.1 M NaClO₄, k(ferricytochrome c+e^?_aq)=(2.4±0.4)·10¹⁰ M^?1·s^?1. In contrast, k(ferricytochrome c+H) is insensitive to ionic strength. Time resolution of three spectral stages has been accomplished. The primary spectrum is the first observable spectrum detectable after irradiation and is formed in a second-order process. Its rate of formation is indisting-uishable from the rate of disappearance of the electron spectrum. The secondary spectrum is generated in a true first order intramolecular process, k(p→s)=(1.2±0.1)·10⁵ s^?1. The tertiary spectrum is also generated in a true first-order process, k(s→t)=(1.3±0.2)·10² s^?1. The specific rates of both transformations are independent of the wavelength of measurement. The tertiary spectrum, observable 50 ms after initial reaction and remaining unchanged thereafter for at least 1 s, shows that relaxed ferrocytochrome c is the only detectable product. This product is not autoxidizable, as expected for native reduced enzyme. It is more probable that the intramolecular changes responsible for the p→s and s→t spectral transformations involve the influence of conformational relaxation of ferrocytochrome c upon electronic energy states then that they are intramolecular transmission of reducing equivalents from primary sites of electron attachment.