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.     

A new redox loop formality involving metal-catalysed hydroxide-ion translocation. A hypothetical Cu loop mechanism for cytochrome oxidase

A new hypothetical type of redox loop is described, which translocates hydroxide instead of protons. Conventional protonmotive redox loops use carriers of protons with electrons (e.g. QH2/Q systems) to couple electron transfer to the translocation of protons. The putative hydroxidemotive redox loop uses carriers of hydroxide ions against electrons (e.g. transition-metal centres) to couple electron transfer to the translocation of hydroxide ions. This simple idea leads to the proposal of a hydroxidemotive Cu loop mechanism that may possibly be applicable to the CuA or CuB centre of cytochrome oxidase, and might thus account for the coupling of electron transfer to net proton translocation in that osmoenzyme.     

Ultrafast haem-haem electron transfer in cytochrome c oxidase.

Electron transfer between the redox centres is essential for the function of the haem-copper oxidases. To date, the fastest rate of electron transfer between the haem groups has been determined to be ca. 3 x 10(5) s(-1). Here, we show by optical spectroscopy that about one half of this electron transfer actually occurs at least three orders of magnitude faster, after photolysis of carbon monoxide from the half-reduced bovine heart enzyme. We ascribe this to the true haem-haem electron tunnelling rate between the haem groups.     

The loneliness of the electrons in the bc1 complex

A stochastic approach based on Gillespie algorithm is particularly well adapted to describe the time course of the redox reactions that occur inside the respiratory chain complexes because they involve the motion of single electrons between individual unique redox centres of a given complex and not populations of electrons and redox centres as usually considered in ordinary differential equations. In this way we approach the molecular functioning of the bc(1) complex based on its known crystallographic structure and the rate constants of electron tunnelling derived from the Moser and Dutton phenomenological equation. The main features of our simulations are the dominant and robust emergence of a Q-cycle mechanism and the near absence of short-circuits in the normal functioning of the bc(1) complex. Thus, in our paper, the Mitchell Q-cycle no longer appears as an a priori hypothesis but arises out of the bc(1) complex structure and of the kinetic laws of redox reactions.     

Colworth Medal Lecture. Enzymes in the quantum world

Studies on those enzymes and electron-transfer proteins involved in the catabolism of 'C1' substrates in methylotrophic bacteria have provided a wealth of information concerning the transfer of electrons and hydrogen by quantum tunnelling mechanisms. With regard to H-transfer, studies with MADH have provided the first example of ground-state tunnelling of hydrogen driven by the natural, thermally activated, low-frequency motions of the enzyme molecule. Subsequent studies with related enzymes (e.g. TMADH and bacterial sarcosine oxidase) and with thermophilic alcohol dehydrogenase suggest that vibrationally assisted tunnelling of hydrogen may be more widespread than originally assumed. Our studies of electron transfer in TMADH and ETF have established a role for large-scale protein dynamics in interprotein electron transfer, and have made a contribution to the ongoing debate concerning the mechanism of amine oxidation by enzymes. Moreover, our work has identified a hitherto unknown mechanism for the control of electron density in reduced flavin that influences the rate of electron transfer between redox centres within a protein molecule. Despite this progress, however, many questions still remain to be resolved. With the development of more sophisticated experimental techniques (and also continued financial support from the funding agencies!), the mechanistic uncertainties surrounding the quantum mechanical transfer of electrons and hydrogen in biological molecules should be transmogrified into the certainties one more readily acquaints with the classical world.     

The membrane-extrinsic domain of cytochrome b(558/566) from the archaeon Sulfolobus acidocaldarius performs pivoting movements with respect to the membrane surface

The orientation of the membrane-attached cytochrome b(558/566)-haem with respect to the membrane was determined by electron paramagnetic resonance spectroscopy on two-dimensionally ordered oxidised membrane fragments from Sulfolobus acidocaldarius. Unlike the other redox centres in the membrane, the cytochrome b(558/566)-haem was found to cover a range of orientations between 25 degrees and 90 degrees. The described results are reminiscent of those obtained on the Rieske cluster of bc complexes and indicate that the membrane-extrinsic domain of cytochrome b(558/566) can perform pivoting motion between two extreme positions. Such a conformational flexibility is likely to play a role in electron transfer with its redox partners.     

Intramolecular electron transfer in [4Fe-4S] proteins: estimates of the reorganization energy and electronic coupling in Chromatium vinosum ferredoxin

The semi-classical electron transfer theory has been very successful in describing reactions occurring in biological systems, but the relevant parameters in the case of iron-sulfur proteins remain unknown. The recent discovery that 2[4Fe-4S] proteins homologous to Chromatium vinosum ferredoxin contain clusters with different reduction potentials now gives the opportunity to study the dependence of the intramolecular electron transfer rate between these clusters as a function of the driving force. This work shows how decreasing the reduction potential difference between the clusters by site-directed mutagenesis of C. vinosum ferredoxin modifies the rate of electron hopping between the two redox sites of the protein by measuring the line broadening of selected 1H NMR signals. Beside the shifts of the reduction potentials, no signs of large structural changes or of significant alterations of the intrinsic kinetic parameters among the different variants of C. vinosum ferredoxin have been found. A reorganization energy of less than 0.5 eV was deduced from the dependence of the electron transfer rates with the reduction potential difference. This small value is associated with a weak electronic coupling between the two closely spaced clusters. This set of parameters, determined for the first time in an iron-sulfur protein, may help to explain how efficient vectorial electron transfer occurs with a small driving force in the many enzymatic systems containing a 2[4Fe-4S] domain.     

Structure and electrochemistry of oxidoreductases

The principles on which Nature has developed multifunctional redox centres, covering a large range of potentials, protected from water and oxygen and surrounded by highly specific proteins, are demonstrated. Structures and accessibilities of the active sites of iron-sulphur proteins, sulphur proteins, flavoproteins, cytochromes and copper proteins are correlated with their possibilities and modes of electron exchange with natural partners, artificial mediators and (modified) electrodes. The participation of charge-transfer and tunnelling processes in electron transport is demonstrated, and a suitable relative orientation of the partners is recognized as one of the most important requirements for electrochemical communication between large molecules and electrodes. The use of specifically modified electrode surfaces, for example those based on electroconductive polymers, is proposed as one of the aspects of future developments for direct electron transfer to proteins.     

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.     

Extensive conformational sampling in a ternary electron transfer complex

Here we report the crystal structures of a ternary electron transfer complex showing extensive motion at the protein interface. This physiological complex comprises the iron-sulfur flavoprotein trimethylamine dehydrogenase and electron transferring flavoprotein (ETF) from Methylophilus methylotrophus. In addition, we report the crystal structure of free ETF. In the complex, electron density for the FAD domain of ETF is absent, indicating high mobility. Positions for the FAD domain are revealed by molecular dynamics simulation, consistent with crystal structures and kinetic data. A dual interaction of ETF with trimethylamine dehydrogenase provides for dynamical motion at the protein interface: one site acts as an anchor, thereby allowing the other site to sample a large range of interactions, some compatible with rapid electron transfer. This study establishes the role of conformational sampling in multi-domain redox systems, providing insight into electron transfer between ETFs and structurally distinct redox partners.     

The flitting of electrons in complex I: A stochastic approach

A stochastic approach based on the Gillespie algorithm is particularly well adapted to describe the time course of the redox reactions that occur inside the respiratory chain complexes because they involve the motion of single electrons between the individual unique redox centres of a given complex. We use this approach to describe the molecular functioning of the peripheral arm of complex I based on its known crystallographic structure and the rate constants of electron tunnelling derived from the Moser and Dutton phenomenological equations. There are several possible electrons pathways but we show that most of them take the route defined by the successive sites and redox centres: NADH⁺ site – FMN – N3 – N1b – N4 – N5 – N6a – N6b – N2 – Q site. However, the electrons do not go directly from NADH towards the ubiquinone molecule. They frequently jump back and forth between neighbouring redox centres with the result that the net flux of electrons through complex I (i.e. net number of electrons reducing a ubiquinone) is far smaller than the number of redox reactions which actually occur. While most of the redox centres are reduced in our simulations the degree of reduction can vary according to the individual midpoint potentials. The high turnover number observed in our simulation seems to indicate that, in the whole complex I, one or several slower step(s) follow(s) the redox reactions involved in the peripheral arm. It also appears that the residence time of FMNH? and SQ? (possible producers of ROS) is low (around 4% and between 1.6% and 5% respectively according to the values of the midpoint potentials). We did not find any evidence for a role of N7 which remains mainly reduced in our simulations. The role of N1a is complex and depends upon its midpoint potential. In all cases its presence slightly decreases the life time of the flavosemiquinone species. These simulations demonstrate the interest of this type of model which links the molecular physico-chemistry of the individual redox reactions to the more global level of the reaction, as is observed experimentally.     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: the phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster F(X)

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A(1), the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F(X) and the phylloquinone bound to either the PsaA (A(1A)) or the PsaB (A(1B)) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F(A) and F(B). The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A(1)) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F(X). A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A(1B) quinone and slightly endergonic, in the case of the A(1A) quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A(0) on both electron transfer branches and the reduction of F(A) by F(X).     

Tunnelling of electrons in biological processes

A new mechanism of energy conversion in biological systems (particularly for phosphorylation on intracellular membranes) is proposed. This mechanism involves electron tunnelling accompanied by relaxation type conformational changes in enzyme macromolecules. The membrane potential may be considered as a regulator of this process.Electron tunnelling is the most important mechanism of electron transfer between electron carriers in electron transport chains of chloroplasts and mitochondria. The requirements of energy balance are met due to excitation or changing of the normal vibrations of carriers or molecules in the medium.     

Structure and reaction mechanisms of multifunctional mitochondrial cytochrome bc1 complex.

The cytochrome bc1 complex from bovine heart mitochondria is a multi-functional enzyme complex. In addition to electron and proton transfer activity, the complex also processes an activatable peptidase activity and a superoxide generating activity. The crystal structure of the complex exists as a closely interacting functional dimer. There are 13 transmembrane helices in each monomer, eight of which belong to cytochrome b, and five of which belong to cytochrome c1, Rieske iron-sulfur protein (ISP), subunits 7, 10 and 11, one each. The distances of 21 A between bL heme and bH heme and of 27 A between bL heme and the iron-sulfur cluster (FeS), accommodate well the observed fast electron transfers between the involved redox centers. However, the distance of 31 A between heme c1 and FeS, makes it difficult to explain the high electron transfer rate between them. 3D structural analyses of the bc1 complexes co-crystallized with the Qu site inhibitors suggest that the extramembrane domain of the ISP may undergo substantial movement during the catalytic cycle of the complex. This suggestion is further supported by the decreased in the cytochrome bc1 complex activity and the increased in activation energy for mutants with increased rigidity in the neck region of ISP.     

Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: structural basis for substrate oxidation and electron transfer.

Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 A resolution. It comprises two domains. The N-terminal domain has a beta-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function. The C-terminal domain is an alpha-helical type I cytochrome c with His(608) and Met(647) as heme-iron ligands. This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c. The shortest distance between pyrroloquinoline quinone and heme c is 12.9 A, one of the longest physiological edge-to-edge distances yet determined between two redox centers. A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent.     

Ultra-high redox enzyme signal transduction using highly ordered carbon nanotube array electrodes

We report on a highly ordered array of carbon nanotubes (CNTs) that serves as a universally direct nanoelectrode interface for redox proteins and provides an efficient conduit for electron transfer. The site-selective, covalent docking of the enzyme glucose oxidase (GO(x)) on the CNT tips is found to have a marked effect on enhancing electron transfer properties, as measured by cyclic voltammetry. A unimolecular electron transfer rate of 1500 s(-1) has been measured for this system, a value exceeding the rate of oxygen reduction by glucose oxidase. Furthermore, the redox enzyme-CNT array conjugate can be utilized as a quantitative, substrate-specific biosensor.     

Coupled motions direct electrons along human microsomal P450 Chains

Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR) to all microsomal cytochrome P450s (CYPs). Our hypothesis is that tight coupling of motion with enzyme chemistry can signal "ready and waiting" states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals "ready and waiting" conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron transfer along complex redox chains.     

Proton-coupled electron transfer: the mechanistic underpinning for radical transport and catalysis in biology

Charge transport and catalysis in enzymes often rely on amino acid radicals as intermediates. The generation and transport of these radicals are synonymous with proton-coupled electron transfer (PCET), which intrinsically is a quantum mechanical effect as both the electron and proton tunnel. The caveat to PCET is that proton transfer (PT) is fundamentally limited to short distances relative to electron transfer (ET). This predicament is resolved in biology by the evolution of enzymes to control PT and ET coordinates on highly different length scales. In doing so, the enzyme imparts exquisite thermodynamic and kinetic controls over radical transport and radical-based catalysis at cofactor active sites. This discussion will present model systems containing orthogonal ET and PT pathways, thereby allowing the proton and electron tunnelling events to be disentangled. Against this mechanistic backdrop, PCET catalysis of oxygen-oxygen bond activation by mono-oxygenases is captured at biomimetic porphyrin redox platforms. The discussion concludes with the case study of radical-based quantum catalysis in a natural biological enzyme, class I Escherichia coli ribonucleotide reductase. Studies are presented that show the enzyme utilizes both collinear and orthogonal PCET to transport charge from an assembled diiron-tyrosyl radical cofactor to the active site over 35A away via an amino acid radical-hopping pathway spanning two protein subunits.     

FesM, a membrane iron-sulfur protein, is required for cyclic electron flow around photosystem I and photoheterotrophic growth of the cyanobacterium Synechococcus sp. PCC 7002

While it is known that cyclic electron flow around photosystem I is an important pathway of photosynthetic electron transfer for converting light energy to chemical energy, some components of cyclic electron flow remain to be revealed. Here, we show that fesM, encoding a novel membrane iron-sulfur protein, is essential to cyclic electron flow in the cyanobacterium Synechococcus sp. PCC 7002. The FesM protein is predicted to have a cAMP-binding domain, an NtrC-like domain, a redox sensor motif, and an iron-sulfur (4Fe-4S) motif. Deletion of fesM (fesM-D) led to an inability for Synechococcus cells to grow in the presences of glycerol and 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Photoheterotrophic growth was restored by a complete fesM gene present on a replicable plasmid. A mutant fesM gene encoding a truncated FesM protein lacking the cAMP domain failed to restore the phenotype, suggesting this domain is important to the function of FesM. Measurements of reduction of P700(+) and the redox state of interphotosystem electron carriers showed that cells had a slower rate of respiratory electron donation to the interphotosystem electron transport chain, and cyclic electron flow around photosystem I in fesM-D was impaired, suggesting that FesM is a critical protein for respiratory and cyclic electron flow. Phosphatase fusion analysis showed that FesM contains nine membrane-spanning helices, and all functional domains of FesM are located on the cytoplasmic face of the thylakoid membranes.