SCIENCE
Progress Made in Understanding Biological Electron Transfer Chemists close in on how factors such as electron donor-acceptor separation and energetics of reaction affect transfer rate Rudy M. Baum, C&EN San Francisco
Until only a few years ago, some pretty basic aspects of biological electron transfer reactions were a matter of dispute. For instance, some researchers believed that the maximum distance across that an electron could move from a donor to an acceptor under physiological conditions was on the order of five or so angstroms. Others thought, and subsequently proved, that significant electron transfer rates occur under such conditions across distances of 10 to 20 A. All the details of biological electron transfer reactions have by no means been sorted out. It is, in fact, a field in intellectual ferment, as was demonstrated at the American Chemical Society's national meeting last month in Denver by a symposium sponsored by the Division of Inorganic Chemistry. Bioinorganic chemists are developing elegant experimental and theoretical techniques to answer the fundamental questions they have posed about this critical biological process. Robert A. Scott, an assistant professor of chemistry at the University of Illinois, Urbana-Champaign, organized the symposium. Scott tells C&EN that the intense interest in biological electron transfer stems from its being "conceptually a very simple process—just getting an electron from point A to point B. Therefore, one can think about it in straightforward terms. However, we
know so little about the mechanism of these electron transfer reactions that there are still many fundamental discoveries to be made." Scott, who will be moving to the University of Georgia this fall, also notes that the rapid progress in the field over the past few years is at least partially the result of the personalities of the chemists involved in the research. "The main players are not only first-rate scientists, but are so outgoing and personable that fertile exchange of results and ideas is possible," Scott says. This view was borne out by the symposium talks, many of which were marked by a bantering camaraderie and friendly one-upsmanship that isn't common in such forums. That electron transfer can occur across relatively long distances in biological systems was first demonstrated about three years ago by four independent research groups (C&EN, Dec. 24,1984, page 24). California Institute of Technology
Hoffman: effect of phenylalanine
chemistry professor Harry B. Gray and Rutgers University chemistry professor Stephan S. Isied each hit upon the idea of studying longrange electron transfer by attaching a ruthenium species to surface histidine residues of metalloproteins such as myoglobin or cytochrome c. Northwestern University chemistry professor Brian M. Hoffman and University of Rochester chemistry professor George L. McLendon each developed metalloprotein systems in which a zinc-porphyrin replaces the natural iron-containing heme group to study photo-induced longrange electron transfer. The four research groups clearly demonstrated that, although the rate of electron transfer in these systems is strongly dependent on the distance between electron donor and acceptor, transfer can occur between redox centers separated by 20 A or more. Since then, Gray, Isied, Hoffman, and McLendon, as well as a number of other chemists such as Scott, have worked to clarify the distance dependence of electron transfer rates. They also have worked to elucidate the effects on electron transfer rates of factors such as the orientation of donor and acceptor groups relative to each other, the energetics of the reaction, and the nature of the protein medium between the donor and acceptor. McLendon, this year's recipient of the ACS Award in Pure Chemistry, described studies of electron transfer between the metalloproteins cytochrome b^ and cytochrome c, and between cytochrome c and cytochrome c peroxidase designed to probe how energy considerations affect electron transfer rates. Working with tight-binding, physiological electron donor-acceptor pairs such as these establishes fixed distances for electron transfer; in the May 4, 1987 C&EN
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Science
Gray: studies on
myoglobin
cytochrome b 5 -cytochrome c com plex, molecular modeling studies suggest that the distance between heme irons is about 15 A; in the cytochrome c-cytochrome c peroxi dase complex, about 22 A. With such fixed distances between donor and acceptor, McLendon and coworkers can study the effect of varying oth er parameters on the electron trans fer reaction. In biological electron transfer cou ples such as these, the free energy change for the electron transfer re action is generally quite small. One question McLendon addressed was whether increasing the free energy difference—that is, the thermody namic driving force—would affect the rates of electron transfer. Mc Lendon varies the driving force by changing the heme group in cyto chrome c. For instance, the iron can be removed yielding porphyrin cy tochrome c; alternatively a zinc can replace the iron. In either case, the cytochrome c redox potential is changed. What the scientists find is that increasing the free energy dif ference does increase the rate of the transfer reaction over a wide range. This implies, McLendon says, that substantial reorganization of the complex must occur to accommo date the charge transfer and that the energy required for this reor ganization represents a significant barrier to electron transfer. To investigate the reorganization 20
May 4, 1987 C&EN
process, the Rochester chemists used site-directed mutagenesis to change amino acid residues on the surface of cytochrome c that likely are in volved in the binding of cytochrome c to either cytochrome b5 or cyto chrome c peroxidase. They find, for instance, that certain changes that result in less tightly bound complexes increase the rate of elec tron transfer between the proteins. This suggests to McLendon that the nature of the interface in electron donor-acceptor complexes has an ef fect on the electron transfer reac tion. It also suggests to him that not only the rate of electron transfer but the specificity of that transfer, which is largely a function of donoracceptor binding, has been a factor in the evolution of these proteins. "Molecules of this kind not only have to get electrons someplace at an appropriate rate, they have to make sure those electrons get to the right place," McLendon says, and his research suggests that there is a trade-off between the two consid erations. Hoffman described experiments with cytochrome c-cytochrome c per oxidase complexes that suggest that amino acid residues lying in the path of the transferring electron can affect the rate of that transfer.
suggests that a particular phenylal anine residue in cytochrome c might be involved in this "docking" in teraction. Theorists have suggested that aromatic residues such as phe nylalanine, with their delocalized π electrons, might also play a role in facilitating electron transfer through proteins. Working in col laboration with chemists A. Grant Mauk and Michael Smith at the Uni versity of British Columbia, who used site-directed mutagenesis to replace the phenylalanine with oth er amino acid residues, Hoffman de termined that this residue has a dra matic effect on the rate of the back electron transfer reaction but little effect on the forward reaction. "These experiments demonstrate the exquisite sensitivity of the electron transfer process when it is in the physiological direction," Hoffman says. Isied described experiments on electron transfer in model peptide systems. He also discussed an ex periment that suggests that electron transfer in cytochrome c is direc tional and that this directionality is mediated by protein conformational changes. Rather than work with pro tein complexes, Isied introduces a new redox center onto the surface of a protein and follows electron
Molecules such as cytochrome not only have to get electrons someplace at an appropriate rate, they have to make sure those electrons get to therightplace Hoffman and coworkers replace the cytochrome c peroxidase heme group with a zinc-porphyrin group. Photoexcitation produces the zinc triplet state, which transfers an electron to the heme iron in cytochrome c. In a subsequent, thermal reaction, which is analogous to the physiological electron transfer reaction, the re duced iron in cytochrome c trans fers an electron back to the zinc in cytochrome c peroxidase. No crystal structure has been de termined for this or any other elec tron transfer protein complex. How ever, computer modeling of the in teraction between the two proteins
transfer between it and the natural redox center of the protein. In pre vious studies of cytochrome c, Isied and coworkers used pentaammineruthenium as that redox center. They established a rate of about 50 per second for transfer of an electron across about 14 A from the attached ruthenium(II) to the heme iron(III) inside the cytochrome c molecule. Because this rate of electron trans fer is on the same time scale as conformational changes of the pro tein, the chemists decided to probe which of the two processes repre sents the rate-limiting step. To do this, they replace one of the ruthe-
nium ammonia ligands with isonicotinamide, thereby "tuning" the re dox potential of the ruthenium such that the direction of electron trans fer is reversed. That is, the transfer occurs from the heme iron(II) to the surface ruthenium(III). What Isied finds is a dramatic, five orders of magnitude decrease in the rate of electron transfer in this system. The results suggest to Isied that this electron transfer pathway in cyto chrome c is "gated," probably by conformational changes in the pro tein that occur upon oxidation or reduction of the protein's heme iron. Gray also uses ruthenium attached to surface histidine residues of a protein to study long-range elec tron transfer. He described a number of experiments involving myoglo bin, which has four surface histi dine residues available for rutheni um attachment. The Caltech chem ists work only with myoglobin molecules to which a single ruthe nium has been attached. These pro vide electron transfer distances of 13, 19, 20, and 22 A. They have studied electron transfer in each of these modified myoglobins in ef forts to better understand the dis tance dependence of transfer rates and the effects of intervening me dium. However, in his talk, Gray focused primarily on studies of elec tron transfer across the 13-À gap that were designed to elucidate the effect of driving force and reorganization energy on transfer rates. Gray varies the driving force in two ways. One is by replacing the heme iron in myoglobin with palladium to obtain a driving force of 1.9 eV when the acceptor is pentaammineruthenium. The second is to tune the ruthenium redox potential in much the same way Isied did; Gray replaces one ammonia ligand with pyridine to increase the driving force another 0.23 eV. This strategy allows electron transfer to be studied at four different free energies ranging from essentially zero for the transfer from the heme iron to pentaammineruthenium to greater than 2 eV for transfer from palladium to ruthenium with a pyridine and four ammonia ligands. Gray finds that the rates increase with driving force throughout this series of molecules. This implies,
Chemist David Ν. Beratan (left) of Jet Propulsion Laboratory confers with University of Rochester's McLendon during break in symposium he says, a very large reorganization energy in this system, on the order of 2.5 eV. Gray speculated that in the case of the ruthenated myoglobin molecules, this energy is required for reorganization of solvent water molecules around the reduced ruthenium species. According to Scott, the observations by McLendon and Gray of this "surprisingly large reorganizational barrier to electron transfer" may be the most important new insight that arose from the symposium. Ongoing work in Scott's laboratory likely will shed further light on this question. He and coworkers have attached tris(bipyridine)ruthenium to surface lysine residues of cytochrome c. In this system, there is no interface between proteins to contribute to reorganization energy and the tris(bipyridine)ruthenium should have a negligible reorganizational barrier, Scott says. Therefore, electron transfer should be much faster, and increasing the driving force in Scott's modified cytochrome c system should not produce the same sort of increase in electron transfer rates seen by McLendon and Gray. Other researchers at the symposium presented a variety of experimental and theoretical results. Chemists are probing electron transfer using model systems, other proteins, and site-directed mutagenesis of pro-
teins. Theorists are working to model electron transfer reactions in proteins. One such researcher, James B. Matthew, who is with the central R&D department of Du Pont, created a stir with the video presentation of the results of a molecular dynamics simulation of the docking interaction of cytochrome c and cytochrome b5. The simulation followed the motions of the two protein structures coming together. As they do, the two heme groups of the proteins move from a coplanar orientation at the outset of the simulation to a closer, slightly stacked orientation. The cytochrome c phenylalanine group identified as important in electron transfer reactions swings into a position somewhat between the heme groups. The results, which required several hours of computation on an extremely fast computer, were unexpected and suggested that in the complex between the two proteins, the heme groups are significantly closer to each other than previously thought. This offered a potential explanation for certain anomalous experimental results noted by both Gray and McLendon. During a subsequent break, the two chemists immediately began discussing new experiments based on Matthew's simulation. That same sort of excitement ran through much of the two-day symposium. • May 4, 1987 C&EN
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