Electron transfer in ferredoxin: are tunneling pathways evolutionarily conserved?

A theoretical study of electron transfer (ET) pathways in a recently crystallized Clostridium acidurici ferredoxin is reported. The electronic structure of the protein complex is treated at the semiempirical extended Hückel level, and the tunneling pathways are calculated with the rigorous quantum mechanical method of tunneling currents. The model predicts two pathways between the two [4Fe-4S] cubanes: a strong one running directly from Cys(14) to Cys(43) and a weaker one from Cys(14) via Ile(23) to Cys(18), whereas other amino acids do not play a significant role in the electron tunneling. The cysteine ligands conduct almost all of the current when Ile(23) is mutated to valine in silico, so that there is no appreciable change in the ET rate. The calculated value of the transfer matrix element is consistent with the experimentally determined rate of transfer. Results of the sequence analysis performed on this ferredoxin reveal that Ile(23) is a highly variable amino acid compared with the cubane-ligating cysteine amino acids, even though Ile(23) lies directly between the donor and acceptor complexes. We further argue that the homologous proteins with a [3Fe-4S] cofactor, which does not have one of the four cysteine ligands, use the same tunneling pathways as those in this ferredoxin, on the basis of the high homology as well as the absolute conservation of Cys(14) and Cys(43) which serve as the main tunneling conduit. Our results explain why mutation of amino acids around and between the donor and acceptor cubane clusters, including that of Ile(23), does not appreciably affect the rate of transfer and add support to the proposal that there exist evolutionarily conserved electron tunneling pathways in biological ET reactions.     

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.     

Environmentally coupled hydrogen tunneling. Linking catalysis to dynamics.

Many biological C-H activation reactions exhibit nonclassical kinetic isotope effects (KIEs). These nonclassical KIEs are too large (kH/kD > 7) and/or exhibit unusual temperature dependence such that the Arrhenius prefactor KIEs (AH/AD) fall outside of the semiclassical range near unity. The focus of this minireview is to discuss such KIEs within the context of the environmentally coupled hydrogen tunneling model. Full tunneling models of hydrogen transfer assume that protein or solvent fluctuations generate a reactive configuration along the classical, heavy-atom coordinate, from which the hydrogen transfers via nuclear tunneling. Environmentally coupled tunneling also invokes an environmental vibration (gating) that modulates the tunneling barrier, leading to a temperature-dependent KIE. These properties directly link enzyme fluctuations to the reaction coordinate for hydrogen transfer, making a quantum view of hydrogen transfer necessarily a dynamic view of catalysis. The environmentally coupled hydrogen tunneling model leads to a range of magnitudes of KIEs, which reflect the tunneling barrier, and a range of AH/AD values, which reflect the extent to which gating modulates hydrogen transfer. Gating is the primary determinant of the temperature dependence of the KIE within this model, providing insight into the importance of this motion in modulating the reaction coordinate. The potential use of variable temperature KIEs as a direct probe of coupling between environmental dynamics and the reaction coordinate is described. The evolution from application of a tunneling correction to a full tunneling model in enzymatic H transfer reactions is discussed in the context of a thermophilic alcohol dehydrogenase and soybean lipoxygenase-1.     

Pathways,pathway tubes,pathway docking,and propagators in electron transfer proteins

The simplest views of long-range electron transfer utilize flat one-dimensional barrier tunneling models, neglecting structural details of the protein medium. The pathway model of protein electron transfer reintroduces structure by distinguishing between covalent bonds, hydrogen bonds, and van der Waals contacts. These three kinds of interactions in a tunneling pathway each have distinctive decay factors associated with them. The distribution and arrangement of these bonded and nonbonded contacts in a folded protein varies tremendously between structures, adding a richness to the tunneling problem that is absent in simpler views. We review the pathway model and the predictions that it makes for protein electron transfer rates in small proteins, docked proteins, and the photosynthetic reactions center. We also review the formulation of the protein electron transfer problem as an effective two-level system. New multi-pathway approaches and improved electronic Hamiltonians are described briefly as well.     

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.     

Electron tunneling pathways in proteins: influences on the transfer rate

A strategy for calculating the tunneling matrix element dependence on the medium intervening between donor and acceptor in specific proteins is described. The scheme is based on prior studies of small molecules and is general enough to allow inclusion of through bond and through space contributions to the electronic tunneling interaction. This strategy should allow the prediction of relative electron transfer rates in a number of proteins. It will therefore serve as a design tool and will be explicitly testable, in contrast with calculations on single molecules. As an example, the method is applied to ruthenated myoglobin and the tunneling matrix elements are estimated. Quantitative improvements of the model are described and effects due to motion of the bridging protein are discussed. The method should be of use for designing target proteins having tailored electron transfer rates for production with site directed mutagenesis. The relevance of the technique to understanding certain photosynthetic reaction center electron transfer rates is discussed.     

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.     

Pathways of electron transfer in Escherichia coli DNA photolyase: Trp306 to FADH

We describe the results of a series of theoretical calculations of electron transfer pathways between Trp306 and *FADH. in the Escherichia coli DNA photolyase molecule, using the method of interatomic tunneling currents. It is found that there are two conformationally orthogonal tryptophans, Trp359 and Trp382, between donor and acceptor that play a crucial role in the pathways of the electron transfer process. The pathways depend vitally on the aromaticity of tryptophans and the flavin molecule. The results of this calculation suggest that the major pathway of the electron transfer is due to a set of overlapping orthogonal pi-rings, which starts from the donor Trp306, runs through Trp359 and Trp382, and finally reaches the flavin group of the acceptor complex, FADH.     

Vibrationally enhanced tunneling as a mechanism for enzymatic hydrogen transfer.

We present a theory of enzymatic hydrogen transfer in which hydrogen tunneling is mediated by thermal fluctuations of the enzyme's active site. These fluctuations greatly increase the tunneling rate by shortening the distance the hydrogen must tunnel. The average tunneling distance is shown to decrease when heavier isotopes are substituted for the hydrogen or when the temperature is increased, leading to kinetic isotope effects (KIEs)--defined as the factor by which the reaction slows down when isotopically substituted substrates are used--that need be no larger than KIEs for nontunneling mechanisms. Within this theory we derive a simple KIE expression for vibrationally enhanced ground state tunneling that is able to fit the data for the bovine serum amine oxidase (BSAO) system, correctly predicting the large temperature dependence of the KIEs. Because the KIEs in this theory can resemble those for nontunneling dynamics, distinguishing the two possibilities requires careful measurements over a range of temperatures, as has been done for BSAO.     

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.     

Structural bases of hydrogen tunneling in enzymes: progress and puzzles

Accumulating experimental evidence suggests that the occurrence of hydrogen tunneling is likely to be widespread in enzyme-catalyzed reactions. The realization that hydrogen can transfer via tunneling mechanisms has far-reaching implications for our understanding of enzyme catalysis involving proton, hydride or hydrogen atom transfer reactions. The current status of the field is highlighted by three enzyme systems that have been under intensive study in recent years, including soybean lipoxygenase-1, thermophilic alcohol dehydrogenase and dihydrofolate reductase. Particular attention has been devoted to the issues of whether protein dynamics modulate hydrogen tunneling probability and whether the tunneling process contributes to the catalytic power of enzymes.     

Electron tunneling chains of mitochondria

The single, simple concept that natural selection adjusts distances between redox cofactors goes a long way towards encompassing natural electron transfer protein design. Distances are short or long as required to direct or insulate promiscuously tunneling single electrons. Along a chain, distances are usually 14 A or less. Shorter distances are needed to allow climbing of added energetic barriers at paired-electron catalytic centers in which substrate and the required number of cofactors form a compact cluster. When there is a short-circuit danger, distances between shorting centers are relatively long. Distances much longer than 14 A will support only very slow electron tunneling, but could act as high impedance signals useful in regulation. Tunneling simulations of the respiratory complexes provide clear illustrations of this simple engineering.     

H-tunneling in the multiple H-transfers of the catalytic cycle of morphinone reductase and in the reductive half-reaction of the homologous pentaerythritol tetranitrate reductase

The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperature-dependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the alpha/beta unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperature-dependent reaction rates, again consistent with ground-state tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme.     

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.     

Tunneling nanotubes provide a unique conduit for intercellular transfer of cellular contents in human malignant pleural mesothelioma

Tunneling nanotubes are long, non-adherent F-actin-based cytoplasmic extensions which connect proximal or distant cells and facilitate intercellular transfer. The identification of nanotubes has been limited to cell lines, and their role in cancer remains unclear. We detected tunneling nanotubes in mesothelioma cell lines and primary human mesothelioma cells. Using a low serum, hyperglycemic, acidic growth medium, we stimulated nanotube formation and bidirectional transfer of vesicles, proteins, and mitochondria between cells. Notably, nanotubes developed between malignant cells or between normal mesothelial cells, but not between malignant and normal cells. Immunofluorescent staining revealed their actin-based assembly and structure. Metformin and an mTor inhibitor, Everolimus, effectively suppressed nanotube formation. Confocal microscopy with 3-dimensional reconstructions of sectioned surgical specimens demonstrated for the first time the presence of nanotubes in human mesothelioma and lung adenocarcinoma tumor specimens. We provide the first evidence of tunneling nanotubes in human primary tumors and cancer cells and propose that these structures play an important role in cancer cell pathogenesis and invasion.     

Geometry for the Primary Electron Donor and the Bacteriopheophytin Acceptor in Rhodopseudomonas viridis Photosynthetic Reaction Centers

The tetrapyrrole electron donors and acceptors (bacteriochlorophyll, BCh; bacteriopheophytin, BPh) within the bacterial photosynthetic reaction center (RC) are arranged with a specific geometry that permits rapid (picosecond time scale) electron tunneling to occur between them. Here we have measured the angle between the molecular planes of the bacteriochlorophyll dimer (primary donor), B₂, and the acceptor bacteriopheophytin, H, by analyzing the dichroism of the absorption change associated with H reduction, formed by photoselection with RCs of Rhodopseudomonas viridis. This angle between molecular planes is found to be 60° ± 2. This means that the ultrafast electron tunneling must occur between donors and acceptors that are fixed by the protein to have a noncoplanar alignment. Nearly perpendicular alignments have been determined for other electron tunneling complexes involving RCs. These geometries can be contrasted with models proposed for heme-heme electron transfer complexes, which have emphasized that mutually parallel orientations should permit the most kinetically facile transfers.     

Effect of protein orientation on electron transfer between photosynthetic reaction centers and carbon electrodes

A new type of monolayer of photosynthetic reaction centers (RC) with the primary donor facing the carbon electrode has been constructed using a new bifunctional linker and genetically engineered protein. Comparison of protein in two different orientations with linkers binding to the opposite sides of the protein demonstrates the possibility of utilizing the constructed surfaces as photoelectronic devices. The results show improvement of the electron transfer efficiency when RC is bound with the primary donor (P) facing the electrode (P-side). In either protein orientation, electron transfer within the protein is unidirectional and when applying a voltage RC operates as a photorectifier. Electron transfer between the protein and carbon electrodes in the constructed devices is most likely occurring by tunneling.     

The role of tunneling in enzyme catalysis of C-H activation

Recent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C-H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects.     

The role of tunneling in enzyme catalysis of C-H activation

Recent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C-H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects.     

DNA repair mechanism by photolyase: electron transfer path from the photolyase catalytic cofactor FADH(-) to DNA thymine dimer

Photolyase is an enzyme that catalyses photorepair of thymine dimers in UV damaged DNA by electron transfer reaction. The structure of the photolyase/DNA complex is unknown at present. Using crystal structure coordinates of the substrate-free enzyme from E. coli, we have recently built a computer molecular model of a thymine dimer docked to photolyase catalytic site and studied molecular dynamics of the system. In this paper, we present analysis of the electronic coupling and electron transfer pathway between the catalytic cofactor FADH(-) and the pyrimidine dimer by the method of interatomic tunneling currents. Electronic structure is treated in the extended Hückel approximation. The root mean square transfer matrix element is about 6 cm(-1), which is consistent with the experimentally determined rate of transfer. We find that electron transfer mechanism responsible for the repair utilizes an unusual folded conformation of FADH(-) in photolyases, in which the isoalloxazine ring of the flavin and the adenine are in close proximity, and the peculiar features of the docked orientation of the dimer. The tunneling currents show explicitly that despite of the close proximity between the donor and acceptor complexes, the electron transfer mechanism between the flavin and the thymine bases is not direct, but indirect, with the adenine acting as an intermediate. These calculations confirm the previously made conclusion based on an indirect evidence for such mechanism.