The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions to numerous biological processes, relatively few techniques are available to measure the actual rate of electron transfer between two redox centers in a protein complex. We have recently introduced a new method to study biological electron transfer that utilizes a covalently attached tris(bipyridine)ruthenium group [Ru(ll)]. Several strategies have been developed for the design and synthesis of ruthenium-labeled redox proteins that are optimized for the measurement of inter protein electron transfer. Over twenty singly labeled derivatives of cytochrome c have now been prepared and characterized. One of the most remarkable properties of Ru(ll) is that it can be photoexcited to a metal-to-ligand charge-transfer state, Ru(lI*), which is a strong reducing agent and can rapidly transfer an electron to the heme group Fe(llI) in cytochrome c. Rate constants up to 3 x 10(7) s-1 are observed for derivatives with separations of about 12 Angstroms between the ruthenium and heme groups. We are using this new technique to measure intracomplex electron transfer between cytochrome c and its physiological partners, cytochrome c oxidase, cytochrome c1, cytochrome c peroxidase. The rate constants for these reactions range from 10(4) to over 10(6) s-1 , and are up to three orders of magnitude larger than previous estimates.
The specific aims for the next grant period are to: 1) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c peroxidase that brings together rapid kinetics, site-directed mutagenesis, and X-ray crystallography. The rate constant, reorganization energy, interaction domain, and pathway of each electron transfer step in the mechanism will be determined. 2) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c oxidase. A major goal will be to determine the pathway and kinetics of electron transfer from cytochrome c through Cu-a and heme a to the heme a3--Cu-B binuclear center under coupled turnover conditions. 3) Carry out a detailed study of the electron transfer reaction between cytochrome c and the cytochrome bc1 complex. A major goal will be to determine the pathway and kinetics of electron transfer from the Rieske iron-sulfur center to cytochrome c1 and to cytochrome c.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM020488-27
Application #
2901975
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1976-06-01
Project End
2003-07-31
Budget Start
1999-08-01
Budget End
2000-07-31
Support Year
27
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Arkansas at Fayetteville
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
191429745
City
Fayetteville
State
AR
Country
United States
Zip Code
72701
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
Bhuiyan, A A; Dossey, R; Anderson, T J et al. (2008) Synthesis and Characterization of Ruthenium(II) Phenanthroline Complexes Containing Quaternary Amine Substituents. J Coord Chem 61:2009-2016
Janzon, Julia; Yuan, Quan; Malatesta, Francesco et al. (2008) Probing the Paracoccus denitrificans cytochrome c(1)-cytochrome c(552) interaction by mutagenesis and fast kinetics. Biochemistry 47:12974-84
Rajagukguk, Sany; Yang, Shaoqing; Yu, Chang-An et al. (2007) Effect of mutations in the cytochrome b ef loop on the electron-transfer reactions of the Rieske iron-sulfur protein in the cytochrome bc1 complex. Biochemistry 46:1791-8
Anderson, Thomas J; Scott, Jill R; Millett, Frank et al. (2006) Decarboxylation of 2,2'-bipyridinyl-4,4'-dicarboxylic acid diethyl ester during microwave synthesis of the corresponding trichelated ruthenium complex. Inorg Chem 45:3843-5