Studies of electron transfer proteins. 1. Spectroscopic studies of cytochrome c peroxidase. 2. Mechanistic and computational studies of cytochrome oxidase
Myers, David Wesley
Doctor of Philosophy
We have measured the magnetic circular dichroism of cytochrome c peroxidase and some of its derivative from 250-350 nm. Comparison of the changes observed on conversion to the catalytic intermediate (cytochrome c peroxidase-I) with spectra obtained from horseradish peroxidase and its derivatives and model compounds of protoheme leads us to the conclusion that the observed changes in the magnetic circular dichroism spectra reflect conversion of the heme to the ferryl state. No evidence was found for the modification of tryptophan in cytochrome c peroxidase I. Five programs for the investigation of steady-state kinetics of catalyzed reactions have been developed. Two provide the solution to the steady-state rate equation: the first of these is a straightforward implementation of the rules developed by Chou, and the second is a very efficient procedure for evaluating King-Altman diagrams and can be used for quite large mechanisms. The third is a program which accepts the output of the second and simplifies the equation to one in identical powers of substrate and products. The fourth program provides the numeric solution to a specific mechanism and set of initial conditions; it is well suited to extremely large, complex mechanisms. The fifth program combines the fourth method and a least squares fitting routine to optimize a mechanism to selected data sets. Mathematical criteria, deduced from empirical observations, have been developed that strictly limit the type of steady-state cytochrome c oxidase mechanisms that are practicable. Two current models (Brzezinski and Malmstrom (1986) Proc. Natl. Acad. Sci. USA 83, 4282-4286; Speck et al. (1984) Proc. Nat. Acad. Sci. USA 81, 347-351) are shown to fail these criteria and thus cannot represent the steady-state reaction of oxidase. Models that do satisfy these criteria are discussed.