Mechanism for electron transfer within and between proteins

Examination of a growing range of electron transfer proteins is clarifying what design elements are and are not naturally selected. Intraprotein electron transfer between natural redox centers is generally engineered to be robust and resistant to mutational changes and thermal fluctuations, by using chains of redox centers connected by electron tunneling distances of 14 A or less. This assures that tunneling rates are faster than the typical millisecond bond-breaking at catalytic sites. Interprotein electron transfer addresses the potential problem of slow diffusion by designing attractive docking sites that permit a conformational search for short tunneling distances.     

Darwin at the molecular scale: selection and variance in electron tunnelling proteins including cytochrome c oxidase

Biological electron transfer is designed to connect catalytic clusters by chains of redox cofactors. A review of the characterized natural redox proteins with a critical eye for molecular scale measurement of variation and selection related to physiological function shows no statistically significant differences in the protein medium lying between cofactors engaged in physiologically beneficial or detrimental electron transfer. Instead, control of electron tunnelling over long distances relies overwhelmingly on less than 14 A spacing between the cofactors in a chain. Near catalytic clusters, shorter distances (commonly less than 7 A) appear to be selected to generate tunnelling frequencies sufficiently high to scale the barriers of multi-electron, bond-forming/-breaking catalysis at physiological rates. We illustrate this behaviour in a tunnelling network analysis of cytochrome c oxidase. In order to surmount the large, thermally activated, adiabatic barriers in the 5-10 kcal mol-1 range expected for H+ motion and O2 reduction at the binuclear centre of oxidase on the 10(3)-10(5) s-1 time-scale of respiration, electron access with a tunnelling frequency of 10(9) or 10(10) s-1 is required. This is provided by selecting closely placed redox centres, such as haem a (6.9 A) or tyrosine (4.9 A). A corollary is that more distantly placed redox centres, such as CuA, cannot rapidly scale the catalytic site barrier, but must send their electrons through more closely placed centres, avoiding direct short circuits that might circumvent proton pumping coupled to haems a to a3 electron transfer. The selection of distances and energetic barriers directs electron transfer from CuA to haem a rather than a3, without any need for delicate engineering of the protein medium to 'hard wire' electron transfer. Indeed, an examination of a large number of oxidoreductases provides no evidence of such naturally selected wiring of electron tunnelling pathways.     

Tunneling in PSII.

With available high resolution structures of PSII and a collection of reported redox midpoint potentials for most of the cofactors, it is possible to compare the expected electron tunneling rates with experimental rates to determine which electron transfer reactions are likely to reflect simply engineered electron tunneling, and which are more sophisticated and associated with large product rearrangements or the making and breaking of bonds. Reliable reorganization energies are largely lacking in this photosystem compared to PSI and purple bacteria and contribute about an order of magnitude uncertainty in tunneling rate estimates. Nevertheless it seems clear that as in purple bacterial reaction centers and PSI, with the notable exception of the oxygen evolving center, the majority of electron transfers within PSII are electron-tunneling limited at room temperature. Tunneling simulations also suggest that the short circuit between pheophytin and the adjacent chlorophyll cation may be fast enough to challenge triplet decay as the principle means of charge recombination from Q(A)(-) at room temperature.     

Distance metrics for heme protein electron tunneling

There is no doubt that distance is the principal parameter that sets the order of magnitude for electron-tunneling rates in proteins. However, there continue to be varying ways to measure electron-tunneling distances in proteins. This distance uncertainty blurs the issue of whether the intervening protein medium has been naturally selected to speed or slow any particular electron-tunneling reaction. For redox cofactors lacking metals, an edge of the cofactor can be defined that approximates the extent in space that includes most of the wavefunction associated with its tunneling electron. Beyond this edge, the wavefunction tails off much more dramatically in space. The conjugated porphyrin ring seems a reasonable edge for the metal-free pheophytins and bacteriopheophytins of photosynthesis. For a metal containing redox cofactor such as heme, an appropriate cofactor edge is more ambiguous. Electron-tunneling distance may be measured from the conjugated heme macrocycle edge or from the metal, which can be up to 4.8 A longer. In a typical protein medium, such a distance difference normally corresponds to a approximately 1000 fold decrease in tunneling rate. To address this ambiguity, we consider both natural heme protein electron transfer and light-activated electron transfer in ruthenated heme proteins. We find that the edge of the conjugated heme macrocycle provides a reliable and useful tunneling distance definition consistent with other biological electron-tunneling reactions. Furthermore, with this distance metric, heme axially- and edge-oriented electron transfers appear similar and equally well described by a simple square barrier tunneling model. This is in contrast to recent reports for metal-to-metal metrics that require exceptionally poor donor/acceptor couplings to explain heme axially-oriented electron transfers.     

Electron transfer in proteins: in search of preferential pathways

Electron migration between and within proteins is one of the most prevalent forms of biological energy conversion processes. Electron transfer reactions take place between active centers such as transition metal ions or organic cofactors over considerable distances at fast rates and with remarkable specificity. The electron transfer is attained through weak electronic interaction between the active sites, so that considerable research efforts are centered on resolving the factors that control the rates of long-distance electron transfer reactions in proteins. These factors include (in addition to the distance and nature of the microenvironment separating the reactants) thermodynamic driving force and the configurational changes required upon reaction. Several of these aspects are addressed in this review, which is based primarily on recent work performed by the authors on model systems of blue copper-containing proteins. These proteins serve almost exclusively in electron transfer reactions, and as it turns out, their metal coordination sites are endowed with properties uniquely optimized for their function.     

Biological electron transfer

Many oxidoreductases are constructed from (a) local sites of strongly coupled substrate-redox cofactor partners participating in exchange of electron pairs, (b) electron pair/single electron transducing redox centers, and (c) nonadiabatic, long-distance, single-electron tunneling between weakly coupled redox centers. The latter is the subject of an expanding experimental program that seeks to manipulate, test, and apply the parameters of theory. New results from the photosynthetic reaction center protein confirm that the electronic-tunneling medium appears relatively homogeneous, with any variances evident having no impact on function, and that control of intraprotein rates and directional specificity rests on a combination of distance, free energy, and reorganization energy. Interprotein electron transfer between cytochromec and the reaction center and in lactate dehydrogenase, a typical oxidoreductase from yeast, are examined. Rates of interprotein electron transfer appear to follow intraprotein guidelines with the added essential provision of binding forces to bring the cofactors of the reacting proteins into proximity.     

Electron flow through metalloproteins

Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Understanding the underlying physics and chemistry of these biological electron transfer processes is the goal of much of the work in our laboratories. Employing laser flash-quench triggering methods, we have shown that 20 Å, coupling-limited Fe(II) to Ru(III) and Cu(I) to Ru(III) electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals; and, further, that analysis of these rates suggests that distant donor-acceptor electronic couplings are mediated by a combination of sigma and hydrogen bonds in folded polypeptide structures. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. In recent work, we have found that 20 Å hole hopping through an intervening tryptophan is several hundred-fold faster than single-step electron tunneling in a Re-modified blue copper protein.     

Electrostatic interaction between redox cofactors in photosynthetic reaction centers

Intramolecular electron transfer within proteins is an essential process in bioenergetics. Redox cofactors are embedded in proteins, and this matrix strongly influences their redox potential. Several cofactors are usually found in these complexes, and they are structurally organized in a chain with distances between the electron donor and acceptor short enough to allow rapid electron tunneling. Among the different interactions that contribute to the determination of the redox potential of these cofactors, electrostatic interactions are important but restive to direct experimental characterization. The influence of interaction between cofactors is evidenced here experimentally by means of redox titrations and time-resolved spectroscopy in a chimeric bacterial reaction center (Maki, H., Matsuura, K., Shimada, K., and Nagashima, K. V. P. (2003) J. Biol. Chem. 278, 3921-3928) composed of the core subunits of Rubrivivax gelatinosus and the tetraheme cytochrome of Blastochloris viridis. The absorption spectra and orientations of the various cofactors of this chimeric reaction center are similar to those found in their respective native protein, indicating that their local environment is conserved. However, the redox potentials of both the primary electron donor and its closest heme are changed. The redox potential of the primary electron donor is downshifted in the chimeric reaction center when compared with the wild type, whereas, conversely, that of its closet heme is upshifted. We propose a model in which these reciprocal shifts in the midpoint potentials of two electron transfer partners are explained by an electrostatic interaction between them.     

Photo-induced charge transfer. A critical test of the mechanism and range of biological electron transfer processes.

The vibronic coupling theory of electron tunneling between biomolecules requires that all such tunnelings involve vibronic coupling, finds temperature dependence to tunneling at finite temperatures, and predicts relatively short tunneling distances. This theory might be expected to apply to most electron transfers involved in the membrane-bound electron transfer reactions of photosynthesis and oxidative phosphorylation. This paper calculates the properties of a weak charge-transfer optical absorption band, whose predicted characteristics are a direct and simple consequence of the model that describes vibronically coupled tunneling. The new absorption band provides the basis for a critical experimental test of the constructs and parameters of the tunneling theory. If the tunneling theory is valid, the oscillator strength of such bands will be the most reliable measure of the tunneling matrix element and of the distance between the sites exchanging an electron.     

Locating a Lipid at the Portal to the Lipoxygenase Active Site

Lipoxygenase enzymes initiate diverse signaling pathways by specifically directing oxygen to different carbons of arachidonate and other polyunsaturated acyl chains, but structural origins of this specificity have remained unclear. We therefore determined the nature of the lipoxygenase interaction with the polar-end of a paramagnetic lipid by electron paramagnetic resonance spectroscopy. Distances between selected grid points on soybean seed lipoxygenase-1 (SBL1) and a lysolecithin spin-labeled on choline were measured by pulsed (electron) dipolar spectroscopy. The protein grid was designed by structure-based modeling so that five natural side chains were replaced with spin labels. Pairwise distances in 10 doubly spin-labeled mutants were examined by pulsed dipolar spectroscopy, and a fit to the model was optimized. Finally, experimental distances between the lysolecithin spin and each single spin site on SBL1 were also obtained. With these 15 distances, distance geometry localized the polar-end and the spin of the lysolecithin to the region between the two domains in the SBL1 structure, nearest to E236, K260, Q264, and Q544. Mutation of a nearby residue, E256A, relieved the high pH requirement for enzyme activity of SBL1 and allowed lipid binding at pH 7.2. This general approach could be used to locate other flexible molecules in macromolecular complexes.     

Characterization of Electron Tunneling and Hole Hopping Reactions between Different Forms of MauG and Methylamine Dehydrogenase within a Natural Protein Complex

Respiration, photosynthesis, and metabolism require the transfer of electrons through and between proteins over relatively long distances. It is critical that this electron transfer (ET) occur with specificity to avoid cellular damage, and at a rate that is sufficient to support the biological activity. A multistep hole hopping mechanism could, in principle, enhance the efficiency of long-range ET through proteins as it does in organic semiconductors. To explore this possibility, two different ET reactions that occur over the same distance within the protein complex of the diheme enzyme MauG and different forms of methylamine dehydrogenase (MADH) were subjected to kinetic and thermodynamic analysis. An ET mechanism of single-step direct electron tunneling from diferrous MauG to the quinone form of MADH is consistent with the data. In contrast, the biosynthetic ET from preMADH, which contains incompletely synthesized tryptophan tryptophylquinone, to the bis-Fe(IV) form of MauG is best described by a two-step hole hopping mechanism. Experimentally determined ET distances matched the distances determined from the crystal structure that would be expected for single-step tunneling and multistep hopping. Experimentally determined relative values of electronic coupling (H(AB)) for the two reactions correlated well with the relative H(AB) values predicted from computational analysis of the structure. The rate of the hopping-mediated ET reaction is also 10-fold greater than that of the single-step tunneling reaction despite a smaller overall driving force for the hopping-mediated ET reaction. These data provide insight into how the intervening protein matrix and redox potentials of the electron donor and acceptor determine whether the ET reaction proceeds via single-step tunneling or multistep hopping.     

Theory of long-distance electron transfer in nanoscale biological structures

Macromolecular biological systems accomplishing the directed electron transfer are nano-sized structures. The distance between carrier molecules (cofactors), which represent practically isolated electron localization centers, reaches tens of angstroms. The electron transfer theory based on the concept of delocalized electron states, which is conventionally used in biophysics, is unable to adequately interpret the results of concrete observations in many cases. On the basis of the theory of electronic transitions in the case of localized states, developed in the physics of disorder matter, a mechanism of long distance electron transfer in biological systems is suggested. The molecular relaxation of the microenvironment of electron localization centers that accompanies the electron transfer process is also considered.     

Electron tunneling through proteins

Electron transfer processes are vital elements of energy transduction pathways in living cells. More than a half century of research has produced a remarkably detailed understanding of the factors that regulate these 'currents of life'. We review investigations of Ru-modified proteins that have delineated the distance- and driving-force dependences of intra-protein electron-transfer rates. We also discuss electron transfer across protein-protein interfaces that has been probed both in solution and in structurally characterized crystals. It is now clear that electrons tunnel between sites in biological redox chains, and that protein structures tune thermodynamic properties and electronic coupling interactions to facilitate these reactions. Our work has produced an experimentally validated timetable for electron tunneling across specified distances in proteins. Many electron tunneling rates in cytochrome c oxidase and photosynthetic reaction centers agree well with timetable predictions, indicating that the natural reactions are highly optimized, both in terms of thermodynamics and electronic coupling. The rates of some reactions, however, significantly exceed timetable predictions: it is likely that multistep tunneling is responsible for these anomalously rapid charge transfer events.     

Comparison of reaction centers from Rhodobacter sphaeroides and Rhodopseudomonas viridis: overall architecture and protein-pigment interactions

Photosynthetic reaction centers (RCs) from the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis are protein complexes closely related in both structure and function. The structure of the Rps. viridis RC was used to determine the structure of the RC from Rb. sphaeroides. Small but meaningful differences between the positions of the helices and the cofactors in the two complexes were identified. The distances between helices AL and AM, between BL and BM, and between bacteriopheophytins BPL and BPM are significantly shorter in Rps. viridis than they are in Rb. sphaeroides RCs. There are a number of differences in the amino acid residues that surround the cofactors; some of these residues form hydrogen bonds with the cofactors. Differences in chemical properties and location of these residues account in some manner for the different spectral properties of the two RCs. In several instances, the hydrogen bonds, as well as the apparent distances between the histidine ligands and the Mg atoms of the bacteriochlorophylls, were found to significantly differ from the Rb. sphaeroides RC structure previously described by Yeates et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7993-7997] and Allen et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8487-8491].     

Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II

Four doubly spin-labeled variants of human carbonic anhydrase II and corresponding singly labeled variants were prepared by site-directed spin labeling. The distances between the spin labels were obtained from continuous-wave electron paramagnetic resonance spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of line-shape broadening, and computer simulation of line-shape changes. Distances also were determined by four-pulse double electron-electron resonance. For each variant, at least two methods were applicable and reasonable agreement between methods was obtained. Distances ranged from 7 to 24 A. The doubly spin-labeled samples contained some singly labeled protein due to incomplete labeling. The sensitivity of each of the distance determination methods to the non-interacting component was compared.     

Electron tunneling pathways in proteins

Long-range electronic interactions between electron donors and acceptors in proteins depend on the structure of the intervening polypeptide. Several methods have been developed for calculating these weak couplings. New challenges in protein electron-transfer research include identifying the role of protein dynamics, and characterizing multistep tunneling over very long distances.     

The electron transfer flavoprotein: ubiquinone oxidoreductases

Electron transfer flavoprotein: ubiqionone oxidoreductase (ETF-QO) is a component of the mitochondrial respiratory chain that together with electron transfer flavoprotein (ETF) forms a short pathway that transfers electrons from 11 different mitochondrial flavoprotein dehydrogenases to the ubiquinone pool. The X-ray structure of the pig liver enzyme has been solved in the presence and absence of a bound ubiquinone. This structure reveals ETF-QO to be a monotopic membrane protein with the cofactors, FAD and a [4Fe-4S](+1+2) cluster, organised to suggests that it is the flavin that serves as the immediate reductant of ubiquinone. ETF-QO is very highly conserved in evolution and the recombinant enzyme from the bacterium Rhodobacter sphaeroides has allowed the mutational analysis of a number of residues that the structure suggested are involved in modulating the reduction potential of the cofactors. These experiments, together with the spectroscopic measurement of the distances between the cofactors in solution have confirmed the intramolecular pathway of electron transfer from ETF to ubiquinone. This approach can be extended as the R. sphaeroides ETF-QO provides a template for investigating the mechanistic consequences of single amino acid substitutions of conserved residues that are associated with a mild and late onset variant of the metabolic disease multiple acyl-CoA dehydrogenase deficiency (MADD).     

High throughput engineering to revitalize a vestigial electron transfer pathway in bacterial photosynthetic reaction centers

Photosynthetic reaction centers convert light energy into chemical energy in a series of transmembrane electron transfer reactions, each with near 100% yield. The structures of reaction centers reveal two symmetry-related branches of cofactors (denoted A and B) that are functionally asymmetric; purple bacterial reaction centers use the A pathway exclusively. Previously, site-specific mutagenesis has yielded reaction centers capable of transmembrane charge separation solely via the B branch cofactors, but the best overall electron transfer yields are still low. In an attempt to better realize the architectural and energetic factors that underlie the directionality and yields of electron transfer, sites within the protein-cofactor complex were targeted in a directed molecular evolution strategy that implements streamlined mutagenesis and high throughput spectroscopic screening. The polycistronic approach enables efficient construction and expression of a large number of variants of a heteroligomeric complex that has two intimately regulated subunits with high sequence similarity, common features of many prokaryotic and eukaryotic transmembrane protein assemblies. The strategy has succeeded in the discovery of several mutant reaction centers with increased efficiency of the B pathway; they carry multiple substitutions that have not been explored or linked using traditional approaches. This work expands our understanding of the structure-function relationships that dictate the efficiency of biological energy-conversion reactions, concepts that will aid the design of bio-inspired assemblies capable of both efficient charge separation and charge stabilization.     

Distance-type reaction coordinates for modelling activated processes

A suitably defined distance is the simplest parameter for measuring the difference between two positions, orientations, and/or conformations of a molecular system. Distances also provide a first guess for the reaction coordinates of activated processes. It is shown here that mass-weighted distances possess remarkable mechanical and statistical mechanical properties. They allow us to restrict motions to internal coordinates of a molecule in a simple way where this demand makes sense. Moreover, the computation of free energy changes and rates is facilitated by simple explicit formulae. The numerical treatment of a rate process in a peptide, the ring flip of a phenylalanine, demonstrates the practical application of our results. It also indicates the role of internal friction in macromolecules and the need to consider transmission coefficients.     

Motility patterns of filamentous sulfur bacteria, Beggiatoa spp

The large sulfur bacteria, Beggiatoa spp., live on the oxidation of sulfide with oxygen or nitrate, but avoid high concentrations of both sulfide and oxygen. As gliding filaments, they rely on reversals in the gliding direction to find their preferred environment, the oxygen-sulfide interface. We observed the chemotactic patterns of single filaments in a transparent agar medium and scored their reversals and the glided distances between reversals. Filaments within the preferred microenvironment glided distances shorter than their own length between reversals that anchored them in their position as a microbial mat. Filaments in the oxic region above the mat or in the sulfidic, anoxic region below the mat glided distances longer than the filament length between reversals. This reversal behavior resulted in a diffusion-like spreading of the filaments. A numerical model of such gliding filaments was constructed based on our observations. The model was applied to virtual filaments in the oxygen- and sulfide-free zone of the sediment, which is a main habitat of Beggiatoa in the natural environment. The model predicts a long residence time of the virtual filament in the suboxic zone and explains why Beggiatoa accumulate high nitrate concentrations in internal vacuoles as an alternative electron acceptor to oxygen.