Frank Millett

Frank Millett

Distinguished Professor, Biochemistry

J. William Fulbright College of Arts & Sciences

(CHBC)-Chemistry & Biochemistry

Phone: 479-575-4999

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American Chemical Society
American Society of Biological Chemists
Biophysical Society

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, which contains four redox active metal centers, CuA, heme a, heme a3, and CuB, reduces all the oxygen we breathe to water and uses the energy to pump protons across the mitochondrial membrane.  However, it has not previously been possible to measure the actual rate of electron transfer between key redox centers in the mitochondria because they are simply too fast.  In collaboration with Professor Bill Durham, we have recently introduced a new method to study biological electron transfer that utilizes a tris(bipyridine)ruthenium(II), [Ru(Il)], complex covalently attached to cyt c.  As an example, Ru(II) was specifically attached to the cysteine sulfhydryl group introduced at residue 39 of cytochrome c (Cc) by site directed mutagenesis to form Ru-39-Cc (Figure 1). One of the most remarkable properties of Ru(II) is that it can be excited by a laser flash to the metal-to-ligand charge-transfer state, Ru(II*), which is a strong reducing agent and rapidly transfers an electron to the heme group Fe(III) in Ru-39-Cc) with a rate constant of 6 x 105 s-1.  The heme in Ru-39-cyt c then transfers an electron to CuA in CcO in 10 ms, followed by electron transfer to heme a in 50 ms, and to heme a3 in 1 ms (Figure 1).  A high-yield ruthenium dimer has also been developed to rapidly inject electrons into CcO, allowing measurement of the kinetics of electron transfer and proton release at each step in the oxygen reduction mechanism.

Cytochrome bc1 contains the Rieske iron-sulfur protein (ISP), cyt c1, and two b-type hemes (bL and bH) in the cyt b subunit.  In the Q-cycle mechanism, the complex translocates four protons to the positive side of the membrane per two electrons transferred from ubiquinol to Cc.  A novel shuttle mechanism has been proposed for the ISP during electron transfer.  With the ISP initially in the b state, QH2 in the Qo site transfers an electron to the oxidized [2Fe2S] center.    The ISP then rotates by 57o to the c1 state where reduced [2Fe2S] transfers an electron to cyt c1 (Figure 2).  The second electron is transferred from semiquinone in the Qo site to cyt bL and then to cyt bH and ubiquinone in the Qi site.  We have developed a new ruthenium dimer which binds with high affinity to cytochrome bc1 and can photooxidize cyt c1 within 1 ms.  This new technique has been used to measure the rate constant for electron transfer from the iron-sulfur center to cyt c1 for the first time, and provides critical information on the dynamics of rotation of the iron-sulfur protein (ISP) as it transfers electrons from QH2 in the Qo site to cyt c1 (Figure 2). 


Figure 1. Transfer from Ru-39-Cc to CcO; Figure 2. Electron Transfer in cyt bc1

NIH Postdoctoral Fellow, California Institute of Technology

Ph.D., Columbia University

B.S., University of Wisconsin

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. R.C. Sadoski, D. Zaslavsky, R.B. Gennis, B. Durham and F. Millett, “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).

5. K. Wang, Y. Zhen, L. Geren, L. Ma, S. Ferguson-Miller, B. Durham and F. Millett, “Mutant of the CuA Site in Cytochrome c Oxidase of Rhodobacter sphaeroides: II. Rapid Kinetic Analysis of Electron Transfer,”Biochemistry, 41, 2298-2304 (2002).

6. H. Mei, L. Geren, M.A. Miller, B. Durham and F. Millett, “Role of the Low-Affinity Binding Site in Electron Transfer from Cytochrome c to Cytochrome c Peroxidase,” Biochemistry, 41, 3968-3976 (2002).

7. G. Engstrom, K. Xiao, C.-A. Yu, L. Yu, B. Durham and F. Millett, “Photoinduced Electron Transfer between the Rieske Iron-Sulfur Protein and Cytochrome c1 in the Rhodobacter sphaeroides Cytochrome bc1 Complex: Effects of pH, Temperature, and Driving Force,” J. Biol. Chem., 277, 31072-31078 (2002).

8. F. Millett and B. Durham, “Design of Photoactive Ruthenium Complexes to Study Interprotein Electron Transfer,” Biochemistry, 41, 11315-11324 (2002).

9. K. Xiao, G. Engstrom, S. Rajagukguk, C.-A. Yu, L. Yu, B. Durham and F. Millett, “Effect of Famoxadone on Photoinduced Electron Transfer between the Iron-Sulfur Center and Cytochrome c1 I the Cytochrome bc1 Complex,” J. Biol. Chem. 278, 11419-11426 (2003).

10. G. Engstrom, R. Rajagukguk, A. J. Saunders, C. Patel, S. Rajagukguk, T. Merbitz-Zahradnik, K. Xiao, G. Pielak, B. Trumpower, C.-A. Yu, L. Gu, B. Durham and F. Millett, “Design of a Ruthenium-labeled Cytochrome c Derivative to Study Electron Transfer with the Cytochrome bc1 Complex,” Biochemistry 42, 2816-2824 (2003).

11. J. Qian, D.A. Mills, L. Geren, K. Wang, C.W. Hoganson, B. Schmidt, C. Hiser, G.T. Babcock, B. Durham, F. Millett and S. Ferguson-Miller, “Role of the Conserved Arginine Pair in Proton and Electron Transfer in Cytochrome C Oxidase,” Biochemistry 43 (19) 5748-56, (2004).

12. B. Durham and F. Millett, “Iron: Heme Proteins and Electron Transport (Cytochromes),” Chapter in Encyclopedia of Inorganic Chemistry (2004).

13. F. Millett, and B. Durham, “Kinetics of Electron Transfer within Cytochrome bc1 and Cytochrome bc1 and Cytochrome c,” Photosynthesis Research 82 (1), 1-16, (2004).

14. Zaslavsky D., Sadoski RC, Rajagukguk S, Geren L, Millett F, Durham B, Gennis RB, “Direct measurement of proton release by chotochrome c oxidase in solution during the F?O transition,” Proc. Natl. Acad. Sci. USA, 101 (29), 10544-7, (2004).

15. D.A. Mills, L. Geren, C. Hiser, B. Schmidt, B. Durham, F. Millett and S. Ferguson-Miller, “An Arginine to Lysine Mutation in the Vicinity of the Heme Propionates Affects the Redox Potentials of the Hemes and Associated Electron and Proton Transfer in Cytochrome c Oxidase,” Biochemistry 44(31), 10457-10465, (2005).

16. Anderson, T.J., Scott; J.R.; Millett, F.; Durham, B., “Decarboxylation of 2.2’-Bipyridinyl-4,4’-dicarboxylic Acid Diethyl Ester during Microwave Synthesis of the Corresponding Trichelated Ruthenium Complex,”Inorg. Chem., 45, 3843-3845, (2006).

17. Siletsky, S.A., Han, D., Brand, S., Morgan, J.E., Fabian, M., Geren, L., Millett, F., Durham, B., Konstantinov, A.A., Gennis, R.B., “Single-electron Photoreduction of the PM Intermediate of Cytochrome c Oxidase,” Biochimica et Biophysica Acta, Bioenergetics, 1757(9-10), 1122-1132 (2006).

18. Rajagukguk, Sany; Yang, Shaoqing; Yu, Chang-An; Yu, Linda; Durham, Bill; Millett, Francis. “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 (2007), 46(7), 1791-1798.

19. Brand, S., Rajagukguk, S., Ganesan, K., Geren, L., Fabian, M., Gennis, R., Durham, B., and Millett, F. “A new Ruthenium Complex to Study Single Electron Reduction of the Pulsed OH State of Detergent-Solubilized Cytochrome Oxidase.” Biochemistry (2007) 46 14610-14618.

20. Bhuiyan, A. A., Dossey, R., Anderson, T. J., Millett, F., and Durham, B. “Synthesis and Characterization of Ruthenium(II) Phenanthroline Complexes Containing Quaternary Amine Substituents”Journal of Coordination Chemistry (2008), 61(13), 2009-2016.

21. Janzon, Julia; Yuan, Quan; Malatesta, Francesco; Hellwig, Petra; Ludwig, Bernd; Durham, Bill; Millett, Francis. “Probing the Paracoccus denitrificans cytochrome c1-Cytochrome c552 interaction by mutagenesis and fast kinetics,” Biochemistry (2008) 47(49), 12974-12984.

22. Mills, D.Z.; Xu, Shujuan; Geren, L.; Hiser, C.; Qin, L.; Sharpe, M.A.; McCracken, J.; Durham, B.; Millett, F.; Ferguson-Miller, S. Proton-Dependent Electron Transfer from CuA to Heme a and Altered EPR Spectra in Mutants Close to Heme a of Cytochrome Oxidase. Biochemistry (2008), 47(44), 11499-11509.

23. Millett, Francis; Durham, Bill, “Use of Ruthenium Photooxidation Techniques to Study Electron Transfer in the Cytochrome bc1 Complex” Chapter 5 in Methods in Enzymology, Mitochondrial Electron Transport Complexes and Reactive Oxygen Species. Methods Enzymol. (2009); 456:95-109.

24. Geren, Lois; Durham, Bill; Millett, Francis. “Use of Ruthenium Photoreduction Techniques to Study Electron Transfer in Cytochrome Oxidase” Chapter 28 in Methods in Enzymology, Mitochondrial Electron Transport Complexes and Reactive Oxygen Species. Methods Enzymol. (2009); 456: 507-20.

25. Williams, A.L.; Bhuiyan, A.A.; Turner, M.O.; Millett, F. and Durham, B. “Synthesis and characterization of Cr(III), Mn(II), Co(II), Ni(II), and CuII) complexes with a hexadenate hemi-cage ligand formed with bipyridine. Journal of Coordination Chemistry (2011), 64(1), 48-56

26. Castellani M, Havens J, Kleinschroth T, Millett F, Durham B, Malatesta F, Ludwig B. The acidic domain of cytochrome c(1) in Paracoccus denitrificans, analogous to the acidic subunits in eukaryotic bc(1) complexes, is not involved in the electron transfer reaction to its native substrate cytochrome c(552).Biochim Biophys Acta. (2011) 1807: 1383-1389

27. Durham B, Millett F. Design of photoactive ruthenium complexes to study electron transfer and proton pumping in cytochrome oxidase. Biochim Biophys Acta. (2011) Sep 10. [Epub ahead of print]

28. Havens J, Castellani M, Kleinschroth T, Ludwig B, Durham B, Millett F. Photoinitiated Electron Transfer within the Paracoccus denitrificans Cytochrome bc(1) Complex: Mobility of the Iron-Sulfur Protein Is Modulated by the Occupant of the Q(o) Site. Biochemistry. (2011) Nov 8. [Epub ahead of print]

1985  Burlington Northern Award for Outstanding Research
1985  Arkansas Alumni Award for Outstanding Research
1991  Haliburton Award for Research
2001  Fulbright College Master Researcher Award
2010  Fellow, American Association for the Advancement of Science
2011  Bruker Analytical Science Professorship