Breaking the Q-cycle: finding new ways to study Qo through thermodynamic manipulations

Thirty years ago, Peter Mitchell won the Nobel Prize for proposing how electrical and proton gradients across bioenergetic membranes were the energy coupling intermediate between photosynthetic and respiratory electron transfer and cellular activities that include ATP production. A high point of his thinking was the development of the Q-cycle model that advanced our understanding of cytochrome bc ₁. While the principle tenets of his Q-cycle still hold true today, Mitchell did not explain the specific mechanism that allows the Qo site to perform this Q-cycle efficiently without undue energy loss. Though much speculation on Qo site mode of molecular action and regulation has been introduced over the 30 years after Mitchell collected his Prize, no single mechanism has been universally accepted. The mystery behind the Qo site mechanism remains unsolved due to elusive kinetic intermediates during Qo site electron transfer that have not been detected spectroscopically. Therefore, to reveal the Qo mechanism, we must look beyond traditional steady-state experimental approaches by changing cytochrome bc ₁ thermodynamics and promoting otherwise transient Qo site redox states. Invited paper to special issue “Peter Mitchell 30th anniversary” for JBB.     

Neurotransmitter release at central synapses

Our understanding of synaptic transmission has grown dramatically during the 15 years since the first issue of Neuron was published, a growth rate expected from the rapid progress in modern biology. As in all of biology, new techniques have led to major advances in the cell and molecular biology of synapses, and the subject has evolved in ways (like the production of genetically engineered mice) that could not even be imagined 15 years ago. My plan for this review is to summarize what we knew about neurotransmitter release when Neuron first appeared and what we recognized we did not know, and then to describe how our views have changed in the intervening decade and a half. Some things we knew about synapses--"knew" in the sense that the field had reached a consensus--are no longer accepted, but for the most part, impressive advances have led to a new consensus on many issues. What I find fascinating is that in certain ways nothing has changed--many of the old arguments persist or recur in a different guise--but in other ways the field would be unrecognizable to a neurobiologist time-transported from 1988 to 2003.     

The Importance of Population Growth and Regulation in Human Life History Evolution

Explaining the evolution of human life history traits remains an important challenge for evolutionary anthropologists. Progress is hindered by a poor appreciation of how demographic factors affect the action of natural selection. I review life history theory showing that the quantity maximized by selection depends on whether and how population growth is regulated. I show that the common use of R, a strategy’s expected lifetime number of offspring, as a fitness maximand is only appropriate under a strict set of conditions, which are apparently unappreciated by anthropologists. To concretely show how demography-free life history theory can lead to errors, I reanalyze an influential model of human life history evolution, which investigated the coevolution of a long lifespan and late age of maturity. I show that the model’s conclusions do not hold under simple changes to the implicitly assumed mechanism of density dependence, even when stated assumptions remain unchanged. This analysis suggests that progress in human life history theory requires better understanding of the demography of our ancestors.     

Variable Electron Transfer Pathways in an Amphibian Cryptochrome: TRYPTOPHAN VERSUS TYROSINE-BASED RADICAL PAIRS*

Electron transfer reactions play vital roles in many biological processes. Very often the transfer of charge(s) proceeds stepwise over large distances involving several amino acid residues. By using time-resolved electron paramagnetic resonance and optical spectroscopy, we have studied the mechanism of light-induced reduction of the FAD cofactor of cryptochrome/photolyase family proteins. In this study, we demonstrate that electron abstraction from a nearby amino acid by the excited FAD triggers further electron transfer steps even if the conserved chain of three tryptophans, known to be an effective electron transfer pathway in these proteins, is blocked. Furthermore, we were able to characterize this secondary electron transfer pathway and identify the amino acid partner of the resulting flavin-amino acid radical pair as a tyrosine located at the protein surface. This alternative electron transfer pathway could explain why interrupting the conserved tryptophan triad does not necessarily alter photoreactions of cryptochromes in vivo. Taken together, our results demonstrate that light-induced electron transfer is a robust property of cryptochromes and more intricate than commonly anticipated.     

Electron transport and transmembrane proton transfer in photosynthetic systems of oxygenic type in Silico

Using a mathematical model of light-induced stages of photosynthesis, which takes into account the key stages of pH-dependent regulation on the acceptor and donor sides of PS I, we analyzed electron and proton transport in chloroplasts of higher plants and in cyanobacterial cells. A comparison of computer simulations with experimental data showed that our model adequately described the complex nonmonotonic kinetics of the light-induced redox transients of P₇₀₀. Effects of atmospheric gases (CO₂ and O₂) on the kinetics of photooxidation of P₇₀₀ and generation of the transmembrane pH difference were studied. We also analyzed how cyclic electron transport influenced the kinetics of electron transfer, intrathylakoid pH, and ATP production. Within the framework of our model, we described the time courses of electron flow through PS II and distribution of electron fluxes on the acceptor side of PS I in chloroplasts and in cyanobacteria. It was demonstrated that contributions of cyclic electron transport and electron flow to O₂ (the Mehler reaction) were significant during the initial phase of the induction period, but diminished upon activation of the Calvin-Benson cycle.     

Electron transfer through the acceptor side of photosystem I: Interaction with exogenous acceptors and molecular oxygen

This review considers the state-of-the-art on mechanisms and alternative pathways of electron transfer in photosynthetic electron transport chains of chloroplasts and cyanobacteria. The mechanisms of electron transport control between photosystems (PS) I and II and the Calvin–Benson cycle are considered. The redistribution of electron fluxes between the noncyclic, cyclic, and pseudocyclic pathways plays an important role in the regulation of photosynthesis. Mathematical modeling of light-induced electron transport processes is considered. Particular attention is given to the electron transfer reactions on the acceptor side of PS I and to interactions of PS I with exogenous acceptors, including molecular oxygen. A kinetic model of PS I and its interaction with exogenous electron acceptors has been developed. This model is based on experimental kinetics of charge recombination in isolated PS I. Kinetic and thermodynamic parameters of the electron transfer reactions in PS I are scrutinized. The free energies of electron transfer between quinone acceptors A_1A/A_1B in the symmetric redox cofactor branches of PS I and iron–sulfur clusters F_X, F_A, and F_B have been estimated. The second-order rate constants of electron transfer from PS I to external acceptors have been determined. The data suggest that byproduct formation of superoxide radical in PS I due to the reduction of molecular oxygen in the A1 site (Mehler reaction) can exceed 0.3% of the total electron flux in PS I.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Phylogenetics

The recent rapid expansion in the DNA and protein databases, arising from large-scale genomic and metagenomic sequence projects, has forced significant development in the field of phylogenetics: the study of the evolutionary relatedness of the planet’s inhabitants. Advances in phylogenetic analysis have greatly transformed our view of the landscape of evolutionary biology, transcending the view of the tree of life that has shaped evolutionary theory since Darwinian times. Indeed, modern phylogenetic analysis no longer focuses on the restricted Darwinian–Mendelian model of vertical gene transfer, but must also consider the significant degree of lateral gene transfer, which connects and shapes almost all living things. Herein, I review the major tree-building methods, their strengths, weaknesses and future prospects.     

The role of protein dynamics and thermal fluctuations in regulating cytochrome c/cytochrome c oxidase electron transfer

In this overview we present recent combined electrochemical, spectroelectrochemical, spectroscopic and computational studies from our group on the electron transfer reactions of cytochrome c and of the primary electron acceptor of cytochrome c oxidase, the Cu_A site, in biomimetic complexes. Based on these results, we discuss how protein dynamics and thermal fluctuations may impact on protein ET reactions, comment on the possible physiological relevance of these results, and finally propose a regulatory mechanism that may operate in the Cyt/CcO electron transfer reaction in vivo. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Cytochrome c551 as a model system for protein folding

Considerable progress was made over the last few years in understanding the mechanism of folding of cytochrome c₅₅₁, a small acidic hemeprotein from Pseudomonas aeruginosa. Comparison of our results with those obtained by others on horse heart cytochrome c allows to draw some general conclusions on the structural features that are common determinants in the folding of members of the cytochrome c family.     

Electrostatics of the FeS clusters in respiratory complex I

Respiratory complex I couples the transfer of electrons from NADH to ubiquinone and the translocation of protons across the mitochondrial membrane. A detailed understanding of the midpoint reduction potentials (E_m) of each redox center and the factors which influence those potentials are critical in the elucidation of the mechanism of electron transfer in this enzyme. We present accurate electrostatic interaction energies for the iron-sulfur (FeS) clusters of complex I to facilitate the development of models and the interpretation of experiments in connection to electron transfer (ET) in this enzyme. To calculate redox titration curves for the FeS clusters it is necessary to include interactions between clusters, which in turn can be used to refine E_m values and validate spectroscopic assignments of each cluster. Calculated titration curves for clusters N4, N5, and N6a are discussed. Furthermore, we present some initial findings on the electrostatics of the redox centers of complex I under the influence of externally applied membrane potentials. A means of determining the location of the FeS cofactors within the holo-complex based on electrostatic arguments is proposed. A simple electrostatic model of the protein/membrane system is examined to illustrate the viability of our hypothesis.     

生物工程强化微生物电合成转化CO_2的研究进展

微生物电合成(Microbial electrosynthesis,MES)可直接利用电能驱动微生物还原固定CO_2合成多碳化合物,为可再生新能源转化、精细化学品制备和生态环境保护提供新机遇。但是,微生物吸收胞外电极电子速率慢、产物合成效率低和产品品位不高,限制了MES实现工业化应用。在概述阴极电活性微生物吸收胞外电子的分子机制的基础上,重点综述近5年应用生物工程的理论和技术强化MES用于CO_2转化的策略与研究进展,包括改造和调控胞外电子传递通路和胞内代谢途径以及定向构建有限微生物混合培养菌群三方面,阐明了生物工程可有效突破MES中电子传递慢和可用代谢途径相对单一等瓶颈。针对目前生物工程在改进MES所面临的主要问题,从胞外电子传递机理研究、基因工具箱开发、组学技术与现代分析技术联用等角度展望了今后的研究方向。     

Tracing the trail of protons through complex I of the mitochondrial respiratory chain

Mitochondria are the structures that produce the bulk part of the cellular energy currency ATP, which drives numerous energy requiring processes in the cell. This process involves a series of large enzyme complexes--the respiratory chain--that couples the transfer of electrons to the creation of a concentration gradient of protons across the inner mitochondrial membrane, which drives ATP synthesis. Complex I (or NADH-quinone oxidoreductase) is the largest and by far the most complicated of the respiratory chain enzyme complexes. The molecular mechanism whereby it couples electron transfer to proton extrusion has remained mysterious until very recently. Low-resolution X-ray structures of complex I have, surprisingly, suggested that electron transfer in the hydrophilic arm, protruding into the mitochondrial matrix, causes movement of a coupling rod that influences three putative proton pumps within the hydrophobic arm embedded in the inner mitochondrial membrane. In this Primer, we will briefly introduce the recent progress made in this area and highlight the road ahead that likely will unravel the detailed molecular mechanisms of complex I function.     

The reaction mechanism of Photosystem I reduction by plastocyanin and cytochrome c6 follows two different kinetic models in the cyanobacterium Pseudanabaena sp. PCC 6903

Plastocyanin (Pc) and cytochrome c₆ (Cyt) have been purified to homogeneity from the cyanobacterium Pseudanabaena sp. PCC 6903, which occupies a unique divergent branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. The two metalloproteins have similar molecular masses (9–10 kDa), as well as almost identical isoelectric points (ca. 8) and midpoint redox potentials (ca. 350 mV, at pH 7). Their reaction mechanism of electron transfer to Photosystem I (PS I) has been analyzed by laser-flash absorption spectroscopy. The kinetic traces with Pc correspond to monophasic kinetics, whereas those with Cyt are better fitted to biphasic curves. The observed pseudo first-order rate constant (k_obs) with Pc and that for the slower phase with Cyt exhibit saturation profiles at increasing donor protein concentrations, thereby suggesting that the two metalloproteins are able to form transient complexes with PS I. The ionic strength dependence of the rate constants for complex formation makes evident the electrostatic nature of intermediate complexes. The experimental findings indicate that the PS I reduction kinetics in Pseudanabaena follow a type II mechanism with Pc and a type III mechanism with Cyt, according to the different kinetic models proposed previously [(Hervás M, Navarro JA, Díaz A, Bottin H and De la Rosa MA (1995) Biochemistry 34: 11321–11326)]. From an evolutionary point of view, this reinforces our previous observation that PS I was first adapted to operate efficiently with positively charged Cyt rather than with Pc.     

DNA内电子传递特性的研究进展

评述了近年来对DNA的电子传递特性的研究进展,并介绍了关于DNA电子传递机制及理论的研究结果。同时,结合从无序体系的角度研究DNA内电子传递特性的工作,对围绕DNA内电子传递问题的争论要点以及尚存在的问题进行了探讨。     

Partial conversion of Hansenula polymorpha amine oxidase into a "plant" amine oxidase: implications for copper chemistry and mechanism

The mechanism of the first electron transfer from reduced cofactor to O2 in the catalytic cycle of copper amine oxidases (CAOs) remains controversial. Two possibilities have been proposed. In the first mechanism, the reduced aminoquinol form of the TPQ cofactor transfers an electron to the copper, giving radical semiquinone and Cu(I), the latter of which reduces O2 (pathway 1). The second mechanism invokes direct transfer of the first electron from the reduced aminoquinol form of the TPQ cofactor to O2 (pathway 2). The debate over these mechanisms has arisen, in part, due to variable experimental observations with copper amine oxidases from plant versus other eukaryotic sources. One important difference is the position of the aminoquinol/Cu(II) to semiquinone/Cu(I) equilibrium on anaerobic reduction with amine substrate, which varies from almost 0% to 40% semiquinone/Cu(I). In this study we have shown how protein structure controls this equilibrium by making a single-point mutation at a second-sphere ligand to the copper, D630N in Hansenula polymorpha amine oxidase, which greatly increases the concentration of the cofactor semiquinone/Cu(I) following anaerobic reduction by substrate. The catalytic properties of this mutant, including 18O kinetic isotope effects, point to a conservation of pathway 2, despite the elevated production of the cofactor semiqunone/Cu(I). Changes in kcat/Km[O2] are attributed to an impact of D630N on an increased affinity of O2 for its hydrophobic pocket. The data in this study indicate that changes in cofactor semiquinone/Cu(I) levels are not sufficient to alter the mechanism of O2 reduction and illuminate how subtle features are able to control the reduction potential of active site metals in proteins.     

The Q-cycle reviewed: How well does a monomeric mechanism of the bc1 complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc₁ complex is reviewed. The data strongly support a mechanism in which the Q_o-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron–sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe–2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q_o-site, and the reduced iron–sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c₁ and liberate the H⁺. When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O₂ is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b_L to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b_L to enhance the rate constant. The acceptor reactions at the Q_i-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b₁₅₀ phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed.The mechanism discussed is applicable to a monomeric bc₁ complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b_L hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.     

Coupled electron transfers in artificial photosynthesis

Light-induced charge separation in molecular assemblies has been widely investigated in the context of artificial photosynthesis. Important progress has been made in the fundamental understanding of electron and energy transfer and in stabilizing charge separation by multi-step electron transfer. In the Swedish Consortium for Artificial Photosynthesis, we build on principles from the natural enzyme photosystem II and Fe-hydrogenases. An important theme in this biomimetic effort is that of coupled electron-transfer reactions, which have so far received only little attention. (i) Each absorbed photon leads to charge separation on a single-electron level only, while catalytic water splitting and hydrogen production are multi-electron processes; thus there is the need for controlling accumulative electron transfer on molecular components. (ii) Water splitting and proton reduction at the potential catalysts necessarily require the management of proton release and/or uptake. Far from being just a stoichiometric requirement, this controls the electron transfer processes by proton-coupled electron transfer (PCET). (iii) Redox-active links between the photosensitizers and the catalysts are required to rectify the accumulative electron-transfer reactions, and will often be the starting points of PCET.     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center