Studies on Cytochrome c Peroxidase

Tryptophan-191 .fwdarw. phenylalanine, a proximal-side mutation in yeast cytochrome c peroxidase that strongly affects the kinetics of ferrocytochrome c oxidation

“…Furthermore, the shape of the pH vs CCP activity curve for the mutant is clearly altered. The pH profile for CCP(MI) shown in Figure 6 is nearly identical with that obtained for bakers' yeast CCP (Yonetani & Ray, 1966), with the added feature that no buffer-related discontinuities occur since the assays were performed in a ternary buffer system at constant ionic strength (µ = 50 mM)…”

“…EPR spectra of CCP(MI) and CCP(MI,F191) were obtained at 89 K in order to ascertain the effects of the mutation on the magnetic properties of the ferric heme at cryogenic temperature. It is well-known that ferric CCP undergoes significant spin-state changes when cooled below room temperature, and when observed frozen at 89 K, the EPR and visible absorption spectra are strongly dependent on pH as well (Yonetani et al, 1966;Yonetani, 1971;Hori & Yonetani, 1985). The pH dependence is manifested as a steady high-to low-spin trend between pH 5 and pH 9, with evidence of loss of heme orientation and/or protein denaturation occurring above pH 9 (Hori & Yonetani, 1985).…”

“…When a 0.73 mM solution of ice-cold CCP(MI,F191) was mixed with a 50% molar excess of H2G2 at pH 6 and rapidly frozen, the 89 K EPR spectrum showed nearly complete conversion to a new species with an isotropic signal centered at g = 2.00 (Figure 4). It is notable that the "wings" flanking the g = 2.00 signal, which are characteristic of compound I of bakers' yeast CCP (Yonetani et al, 1966) and CCP(MI) (Figure 4), are essentially missing in the spectrum of the H202-oxidized CCP(MI,F191) mutant. Diminished wings have been observed for the radical signals of the H202-oxidized Phe-51 and Ser-172 CCP mutants previously studied (Fishel et al, 1987;Goodin et al, 1986).…”

Interaction Domain for the Reaction of Cytochrome c with the Radical and the Oxyferryl Heme in Cytochrome c Peroxidase Compound I

“…A number of different techniques have been used to characterize the reaction between cytochrome c and cytochrome c peroxidase Yonetani & Ray, 1966;Kang etal., 1977;Kim etal., 1990;Summers & Erman, 1988;Hazzard etal., 1987Hazzard etal., ,1988aHazzard & Tollin, 1991;Nuevo et al, 1993). The kinetics measured under steadystate conditions are particularly complicated, since at least two complete cycles of complex formation, electron transfer, and product dissociation are involved (Kim etal., 1990;Erman et al, 1991).…”

Crystal structure of cytochrome c peroxidase compound I

“…We hoped that the electronic redistribution accompanying the formation of a radical, especially a positively charged radical, would cause small but detectable positional shifts of nearby atoms with some partial negative charge character. Indeed, such an experiment had been suggested as long ago as 1966 (Yonetani et al, 1966), although the required techniques were not available until recently. Even though collecting a complete set of diffraction data for a stable protein crystal has now become fairly routine, doing the same for a short-lived enzyme intermediate has only recently become feasible with the development of high-speed area detector diffractometers and compatible low-temperature technology (Xuong et al, 1978).…”