CHARACTERIZATION OF RESPIRATORY PROTEIN COMPLEXES

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Using the results from our previous work on yeast cytochrome c peroxidase (CcP) complexes, this proposal reflects the comments of the previous NIH Study Section which recommended a proposal tightly focused on NMR studies of noncovalent CcP/cytochrome c complexes. In this proposal we intend to focus on comparative studies of a nonphysiological type of CcP complex: between CcP and horse cytochrome c; and a physiological type of CcP complex: between CcP and its natural redox partner yeast iso- 1 cytochrome c. This discrimination of complex type is based upon results from our work during the past three years, which segregate themselves along species-specific lines. One of our most interesting results during this time has been the resolution of free and bound ferricytochrome c resonances in NMR spectra of CcP/ferricytochrome c noncovalent complexes. We propose to further characterize these complexes by NMR spectroscopy, elucidate the complex dissociation rate constants and determine the effects on cytochrome c NMR spectra of forming complexes with native, wild-type CcP (a ferric, high-spin heme protein), and as well, with the oxidized intermediates, CcP-I (compound-I) and CcP-II (compound-II), and cyanide-inhibited CcP (CcPCN). NMR detection of effects of complex formation upon CcP will be attempted using CcPCN in combination with ferro- and ferricytochromes c.