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Frank Millett CHEM 243 |
Degrees:
Ph.D., Columbia University, 1970
NIH Postdoctoral Fellow, California Institute of Technology, 1970-72
Burlington Northern Award for Outstanding Research, 1985
Research Interests:
biological electron transfer
Research:
Bioenergetics, Electron Transfer Reactions
Biological electron transfer reactions play an essential role in energy production in all living organisms. Despite the importance of these reactions, it has rarely been possible to measure the actual rate constant of the electron transfer step because it is simply too fast for conventional kinetic techniques. 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 one of the proteins. Novel site-directed mutagenesis and protein engineering strategies have been developed to prepare over twenty different Ru-labeled derivatives of the small heme proteins cytochrome c and cytochrome b5. 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(IM in Ru39-Cc) with a rate constant of 6 x 105 s-1 (Figure 2). Ru-39-Cc was designed to have an efficient pathway for electron transfer from Ru(II*) to the heme consisting of 12 covalent bonds and one hydrogen bond
The reaction between Cc and cytochrome c peroxidase (CcP) has become one of the most attractive systems for investigating fundamental questions about biological electron transfer. High resolution X-ray crystal structures are now available for both Cc and CcP, and for the 1:1 complex between them. Hydrogen peroxide oxidizes the ferric CcP to the CMPI form of the enzyme, which contains an oxyferryl heme, Fe(IV)=O, and a radical cation located on the indole group of Trp-191. CMPI is then sequentially reduced to CMPII and CcP by two molecules of Cc. An important question is whether Cc first reduces the oxyferryl heme or the radical cation. We found that laser excitation of the 1:1 complex between Ru-39-Cc and CMPI resulted in rapid electron transfer from Ru(ll*) to heme c Fe(III), followed by electron transfer from heme c Fe(II) to the radical cation R* with a rate constant of 2 x 106 s-1 (Figure 2). This indicates that the radical cation on Trp-191 is the initial electron entry site, and that electron transfer is over three orders of magnitude faster than previously expected. This electron transfer reaction appears to utilize an efficient pathway that extends from the edge of heme c through CcP residues Ala-194, Ala-193, and Gly-192 to the indolyl radical cation on Trp-191. Photoexcitation with a second laser flash results in electron transfer from heme c to the oxyferryl heme with a rate constant of 5000 s-1. This much slower reaction appears to involve concerted electron and proton transfer to the oxyferryl heme Fe(IV)=O to form Fe(III) + H2O. We now plan to address a number of other important questions about this system. Ultimately, we would like to crystallize a 1:1 complex between an Ru-Cc derivative and CcP. This would allow us to make a unique correlation between structure and kinetics in interprotein electron transfer.
We are also using the Ru photoreduction technique to study electron transfer from Cc to cytochrome oxidase, which uses the electrons to reduce molecular oxygen to water and couples the energy of electron transfer to the formation of a proton gradient across the mitochondrial membrane. We plan to determine the mechanism of electron transfer from Cc to the redox centers in cytochrome oxidase, copper A, heme a, heme a3and copper B. An important question is which electron transfer steps are energy coupled to proton pumping across the membrane.