The broad objective of the proposed research is to develop new chemical modification and cross-linking techniques to study protein structure and function in biological membranes. We have recently prepared 17 different derivatives of cytochrome c in each of which a single lysine amino group has been modified to form an uncharged CF3CO- or CF3PhNHCO-lysine. Enzyme kinetic studies with these derivatives have shown that the interaction domain for both cytochrome c1 and cytochrome oxidase involves the ring of positively charged lysine amino groups surrounding the heme crevice of cytochrome c. This result suggests that cytochrome c must undergo some type of diffusion as it transports electrons from cytochrome c1 to cytochrome oxidase. Our studies are now focused on identifying which specific residues on cytochromes oxidase and c1 are involved in binding cytochrome c. We have recently used the water-soluble carbodiimide EDC to modify three carboxyl groups on cytochrome oxidase that are required for cytochrome c binding. HPLC peptide mapping was used to identify the labeled carboxylates as Asp 112, Glu 114, and Glu 198 of subunit II. Glu 198 is located between cysteine residues 196 and 200 which have been proposed to ligand the EPR-detectable copper. This copper atom thus appears to be located close enough to the cytochrome c binding site to be reduced directly. Chemical modification techniques are proposed to determine where the other electron acceptor, cytochrome a, is located in the oxidase structure. We have recently developed a new general procedure to cross-link any lysine amino group on cytochrome c to its complementary carboxyl group on the redox partner. HPLC peptide mapping will be used to identify the specific carboxyl group that interacts with each lysine. This should help refine our understanding of the surface topology and folding patterns of these proteins. The type of diffusional motion cytochrome c must undergo will be evaluated from the effect of different specific cross-links on the rate of electron transport from cytochrome c1 to cytochrome oxidase. These studies should also indicate whether cytochrome c1 must have some rotational flexibility to transfer electrons from the iron-sulfur protein in the cytochrome bc1 complex to cytochrome c.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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University of Arkansas at Fayetteville
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Millett, Francis; Havens, Jeffrey; Rajagukguk, Sany et al. (2013) Design and use of photoactive ruthenium complexes to study electron transfer within cytochrome bc1 and from cytochrome bc1 to cytochrome c. Biochim Biophys Acta 1827:1309-19
Durham, Bill; Millett, Francis (2012) Design of photoactive ruthenium complexes to study electron transfer and proton pumping in cytochrome oxidase. Biochim Biophys Acta 1817:567-74
Havens, Jeffrey; Castellani, Michela; Kleinschroth, Thomas et al. (2011) Photoinitiated electron transfer within the Paracoccus denitrificans cytochrome bc1 complex: mobility of the iron-sulfur protein is modulated by the occupant of the Q(o) site. Biochemistry 50:10462-72
Castellani, Michela; Havens, Jeffrey; Kleinschroth, Thomas et al. (2011) The acidic domain of cytochrome c? in paracoccus denitrificans, analogous to the acidic subunits in eukaryotic bc? complexes, is not involved in the electron transfer reaction to its native substrate cytochrome c(552). Biochim Biophys Acta 1807:1383-9
Geren, Lois; Durham, Bill; Millett, Francis (2009) Chapter 28 Use of ruthenium photoreduction techniques to study electron transfer in cytochrome oxidase. Methods Enzymol 456:507-20
Millett, Francis; Durham, Bill (2009) Chapter 5 Use of ruthenium photooxidation techniques to study electron transfer in the cytochrome bc1 complex. Methods Enzymol 456:95-109
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