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Project 5. Structural Basis of Oxidative Phosphorylation

(Mack Ivey, Joshua Sakon, Dan Davis, Lois Geren, Bill Durham, Frank Millett)

Mitochondrial oxidative phosphorylation is a fundamental process in biological energy transformation, and disruption of this process leads to serious human health problems, including mitochondrial myopathies, degenerative diseases, and aging. Cytochrome c (Cyt c) is a small heme protein which functions as a mobile shuttle as it transports electrons from the cytochrome bc1 complex to cytochrome c oxidase (CcO). CcO contains four redox active metal centers, CuA, heme a, heme a3, and CuB. However, it has not previously been possible to measure the actual rate of electron transfer between key redox centers in the mitochondria. We introduced a new method to initiate electron transfer by exciting a ruthenium complex with a nanosecond laser flash. One of the most remarkable properties of ruthenium is that it can be photoexcited to a metal?to?ligand charge?transfer state, Ru(II*), which is a strong reducing agent and can rapidly transfer an electron to the heme group Fe(III) in cytochrome c (Figure). This new method was used to show that Cyt c transfers an electron to CuA in 10 ms, followed by electron transfer to heme a in 50 ms, and to heme a3 in 1 ms. However, the mechanism of energy coupling between cytochrome oxidase and ATP synthase is not well understood, particularly regarding the pathway of proton translocation. We are carrying out a multidisciplinary, collaborative approach to this problem, which combines powerful rapid kinetics techniques, bacterial model systems, and structure determination.

Electron Transfer from Ru-39-Cc to Cytochrome Oxidase

References

1. K. Wang, Y. Zhen, R. Sadoski, S. Grinnell, L. Geren, S. Ferguson-Miller, B. Durham and F. Millett, "Definition of the Interaction Domain for Cytochrome c on Cytochrome c Oxidase. II. Rapid Kinetic Analysis of Electron Transfer from Cytochrome c to Rhodobacter sphaeroides Cytochrome Oxidase Surface Mutants", J. Biol. Chem., 274, 38042-50 (1999).
2. H. Tian, R. Sadoski, L. Zhang, C.-A. Yu, B. Durham and F. Millett, "Definition of the Interaction Domain for Cytochrome c on the Cytochrome bc1 Complex: Steady-state and Rapid Kinetic Analysis of Electron Transfer between Cytochrome c and Rhodobacter sphaeroides Cytochrome bc1 Surface Mutants", J. Biol. Chem., 275, 9587-9595 (2000).
3. R. Sadoski, G. Engstrom, H. Tian, L. Zhang, C-A. Yu, L. Yu, B. Durham and F. Millett, "Use of a Photoactivated Ruthenium Dimer Complex to Measure Electron-Transfer between the Rieski Iron-Sulfur Protein and Cytochrome c1 in the Cytochrome bc1 Complex", Biochemistry, 39, 4231-4236 (2000).
4. Sadoski RC, Zaslavsky D, Gennis RB, Durham B, Millett F. Exposure of Bovine Cytochrome c Oxidase to High Triton X-100 or to Alkaline Conditions Causes a Dramatic Change in the Rate of Reduction of Compound F. J Biol Chem. 276 33616-20 (2001).