The energetics and evolution of oxidoreductases in deep time

The core metabolic reactions of life drive electrons through a class of redox protein enzymes, the oxidoreductases. The energetics of electron flow is determined by the redox potentials of organic and inorganic cofactors as tuned by the protein environment. Understanding how protein structure affects oxidation–reduction energetics is crucial for studying metabolism, creating bioelectronic systems, and tracing the history of biological energy utilization on Earth. We constructed ProtReDox ( https://protein-redox-potential.web.app ), a manually curated database of experimentally determined redox potentials. With over 500 measurements, we can begin to identify how proteins modulate oxidation–reduction energetics across the tree of life. By mapping redox potentials onto networks of oxidoreductase fold evolution, we can infer the evolution of electron transfer energetics over deep time. ProtReDox is designed to include user-contributed submissions with the intention of making it a valuable resource for researchers in this field.     

Biological electron tunneling through native protein media

 Nature has engineered a universe of redox proteins to efficiently control the oxidation and reduction of substrates and to convert redox energy into a delocalized transmembrane proton gradient power source. Some rapid physiologically relevant electron transfers are rate limited by electron tunneling. Distance appears to be the principle means naturally selected to control the speed of electron tunneling; free energy and reorganization energy can play important auxiliary roles. Thus, an electron from a biological redox center can tunnel in any direction and is likely to reduce the closest redox center with a favorable free energy. Although it is clearly possible to facilitate electron tunneling by designing covalent bridges in the regions between donors and acceptors, this does not seem to be a strategy that evolution has used. Evolutionary mutagenic adjustment of a bridge-like quality of the amino acid medium may be difficult in the face of heavy selection on the folding, stability and other properties of the protein medium. Repositioning cofactors by even a few angstroms has more profound effects on promoting and retarding rates, independent of the structure of the amino acid medium. Received: 12 January 1997 / Accepted: 5 February 1997     

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.     

Direct and indirect electron transfer between electrodes and redox proteins

The direct electrochemistry of redox proteins has been achieved at a variety of electrodes, including modified gold, pyrolytic graphite and metal oxides. Careful design of electrode surfaces and electrolyte conditions are required for the attainment of rapid and reversible protein-electrode interaction. The electron transfer reactions of more complex systems, such as redox enzymes, are now being examined. The 'well-behaved' electrochemistry of redox proteins can be usefully exploited by coupling the electrode reaction to enzymes for which the redox proteins act as cofactors. In systems where direct electron transfer is very slow, small electron carriers, or mediators, may be employed to enhance the rate of electron exchange with the electrode. The organometallic compound ferrocene and its derivatives have proved particularly effective in this role. A new generation of electrochemical biosensors employs ferrocene derivatives as mediators.     

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.     

Redox equilibria in hydroxylamine oxidoreductase. Electrostatic control of electron redistribution in multielectron oxidative processes

We report results of continuum electrostatics calculations of the cofactor redox potentials, and of the titratable group pK(a) values, in hydroxylamine oxidoreductase (HAO). A picture of a sophisticated multicomponent control of electron flow in the protein emerged from the studies. First, we found that neighboring heme cofactors strongly interact electrostatically, with energies of 50-100 mV. Thus, cofactor redox potentials depend on the oxidation state of other cofactors, and cofactor redox potentials in the active (partially oxidized) enzyme differ substantially from the values obtained in electrochemical redox titration experiments. We found that, together, solvent-exposed heme 1 (having a large negative redox potential) and heme 2 (having a large positive redox potential) form a lock for electrons generated during the oxidation reaction The attachment of HAO's physiological electron transfer partner cytochrome c(554) results in a positive shift in the redox potential of heme 1, and "opens the electron gate". Electrons generated as a result of hydroxylamine oxidation travel to heme 3 and heme 8, which have redox potentials close to 0 mV versus NHE (this result is in partial disagreement with an existing experimental redox potential assignment). The closeness of hemes 3 and 8 from different enzyme subunits allows redistribution of the four electrons generated as a result of hydroxylamine oxidation, among the three enzyme subunits. For the multielectron oxidation process to be maximally efficient, the redox potentials of the electron-accepting cofactors should be roughly equal, and electrostatic interactions between extra electrons on these cofactors should be minimal. The redox potential assignments presented in the paper satisfy this general rule.     

Mechanisms for regulating electron transfer in multi-centre redox proteins.

Protein-mediated electron transfer is a key process in nature. Many of the proteins involved in such electron transfers are complex and contain a number of redox-active cofactors. The very complexity of these multi-centre redox proteins has made it difficult to fully understand the various electron transfer events they catalyse. This is sometimes because the electron transfer steps themselves are gated or coupled to other processes such as proton transfer. However, with the molecular structures of many of these proteins now available it is possible to probe these electron transfer reactions at the molecular level. It is becoming apparent that many of these multi-centre redox proteins have rather subtle and elegant ways for regulating electron transfer. The purpose of this article is to illustrate how nature has used different approaches to control electron transfer in a number of different systems. Illustrative examples include: thermodynamic control of electron transfer in flavocytochromes b(2) and P450 BM3; a novel control mechanism involving calmodulin-binding-dependent electron transfer in neuronal nitric oxide synthase; the probable gating of electron transfer by ATP hydrolysis in nitrogenase; conformational gating of electron transfer in cytochrome cd(1); the regulation of electron transfer by protein dynamics in the cytochrome bc(1) complex; and finally the coupling of electron transfer to proton transfer in cytochrome c oxidase.     

PAS domains: internal sensors of oxygen, redox potential, and light.

PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells.     

PAS Domains: Internal Sensors of Oxygen, Redox Potential, and Light

PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells.     

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 µM. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.     

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.     

Organic cofactors participated more frequently than transition metals in redox reactions of primitive proteins

Protein redox reactions are one of the most basic and important biochemical actions. As amino acids are weak redox mediators, most protein redox functions are undertaken by protein cofactors, which include organic ligands and transition metal ions. Since both kinds of redox cofactors were available in the pre‐protein RNA world, it is challenging to explore which one was more involved in redox processes of primitive proteins? In this paper, using an examination of the redox cofactor usage of putative ancient proteins, we infer that organic ligands participated more frequently than transition metals in redox reactions of primitive proteins, at least as protein cofactors. This is further supported by the relative abundance of amino acids in the primordial world. Supplementary material for this article can be found on the BioEssays website. BioEssays 30:766–771, 2008. © 2008 Wiley Periodicals, Inc.     

Disruption of mitochondrial redox circuitry in oxidative stress

The present review and commentary considers oxidative stress as a disruption of mitochondrial redox circuitry rather than an imbalance of oxidants and antioxidants. Mitochondria contain two types of redox circuits, high-flux pathways that are central to mechanisms for ATP production and low-flux pathways that utilize sulfur switches of proteins for metabolic regulation and cell signaling. The superoxide anion radical (hereafter termed "superoxide", O2*-), a well known free radical product of the high-flux mitochondrial electron transfer chain, provides a link between the high-flux and low-flux pathways. Disruption of electron flow and increased superoxide production occurs due to inhibition of electron transfer in the high-flux pathway, and this creates aberrant "short-circuit" pathways between otherwise non-interacting components. A hypothesis is presented that superoxide is not merely a byproduct of electron transfer but rather is generated by the mitochondrial respiratory apparatus to serve as a positive signal to coordinate energy metabolism. Electron mediators such as free Fe(3+) and redox-cycling agents, or potentially free radical scavenging agents, could therefore cause oxidative stress by disrupting this normal superoxide signal. Methods to map the regulatory redox circuitry involving sulfur switches (e.g., redox-western blotting of thioredoxin-2, redox proteomics) are briefly presented. Use of these approaches to identify sites of disruption in the mitochondrial redox circuitry can be expected to generate new strategies to prevent toxicity and, in particular, promote efforts to re-establish proper electron flow as a means to counteract pathologic effects of oxidative stress.     

Application of electrochemical kinetics to photosynthesis and oxidative phosphorylation: The redox element hypothesis and the principle of parametric energy coupling

It is suggested that the transfer of electrons within the biological electron transfer chain is subject to the laws of electrochemical kinetics, when membrane-bound electron carriers are involved. Consequently, small tightly bound molecular complexes of two or more electron transfer proteins of different redox potential within an energy transducing membrane, which accept electrons from a donor at one membrane surface and donate it to an acceptor at the other, may be regarded as real and functioning molecular redox elements, which convert the free energy of electrons into electrochemical energy. Especially, the transfer of an electron from excited chlorophyll to an electron acceptor can be looked upon as an electrochemical oxidation of excited chlorophyll at such a complex. In this reaction the electron acceptor complex behaves like a polarized electrode, in which the electrochemical potential gradient is provided by a gradient of redox potential of its constituents.Calculations and qualitative considerations show that this concept leads to a consistent understanding of both primary and secondary reactions in photosynthesis (electron capture, delayed light emission, ion transfer, energy conversion) and can also be applied to oxidative phosphorylation. Within the proposed concept, ion transfer and the development of ion gradients have to be considered as results of electrochemical activity—not as intermediates for energy conversion. For energetic reasons, a non steady state, periodic energy coupling mechanism is postulated which functions by periodic changes of the capacity of the (electrochemically) charged energy transducing membrane, during which capacitive surplus energy is released as chemical energy. Energy transducing membranes may thus be considered as electrochemical parametric energy transformers. This concept explains active periodic conformation changes and mechanochemical processes of energy transducing membranes as energetically essential events, which trigger energy conversion according to the principle of variable parameter energy transformers.The electrochemical approach presented here has been suggested and is supported by the observation, that with respect to electron capture and conversion of excitation energy into electrochemical energy, the behaviour of excited chlorophyll at suitable solid state (semiconductor) electrodes is very similar to that of chlorophyll in photosynthetic reaction centers.     

Structure and mechanism of tryptophylquinone enzymes

Tryptophylquinone cofactors are formed by posttranslational modifications that result in the incorporation of two oxygens into a tryptophan side chain, and the covalent cross-linking of that side chain to another amino acid residue. Tryptophylquinone enzymes catalyze the oxidative deamination of primary amines, and utilize other redox proteins as electron acceptors. Mechanistic and structural studies of these enzymes are providing insight into how these enzymes utilize these highly reactive protein-derived quinones in a controlled manner to facilitate biologically important catalytic and electron transfer reactions.     

Exploring amino-acid radical chemistry: protein engineering and de novo design

Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.     

Exploring amino-acid radical chemistry: protein engineering and de novo design

Amino-acid radical enzymes are often highly complex structures containing multiple protein subunits and cofactors. These properties have in many cases hampered the detailed characterization of their amino-acid redox cofactors. To address this problem, a range of approaches has recently been developed in which a common strategy is to reduce the complexity of the radical-containing system. This work will be reviewed and it includes the light-induced generation of aromatic radicals in small-molecule and peptide systems. Natural redox proteins, including the blue copper protein azurin and a bacterial photosynthetic reaction center, have been engineered to introduce amino-acid radical chemistry. The redesign strategies to achieve this remarkable change in the properties of these proteins will be described. An additional approach to gain insights into the properties of amino-acid radicals is to synthesize de novo designed model proteins in which the redox chemistry of these species can be studied. Here we describe the design, synthesis and characteristics of monomeric three-helix bundle and four-helix bundle proteins designed to study the redox chemistry of tryptophan and tyrosine. This work demonstrates that de novo protein design combined with structural, electrochemical and quantum chemical analyses can provide detailed information on how the protein matrix tunes the thermodynamic properties of tryptophan.     

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.     

The Fe and Zn cofactor dilemma

Fe and Zn ions are essential enzymatic cofactors across all domains of life. Fe is an electron donor/acceptor in redox enzymes, while Zn is typically a structural element or catalytic component in hydrolases. Interestingly, the presence of Zn in oxidoreductases and Fe in hydrolases challenge this apparent functional dichotomy. In hydrolases, Fe either substitutes for Zn or specifically catalyzes certain reactions. On the other hand, Zn can replace divalent Fe and substitute for more complex Fe assemblies, known as Fe-S clusters. Although many zinc-binding proteins interchangeably harbor Zn and Fe-S clusters, these cofactors are only sometimes functional proxies.     

Photosystem II: the engine of life

Photosystem II (PS II) is a multisubunit membrane protein complex, which uses light energy to oxidize water and reduce plastoquinone. High-resolution electron cryomicroscopy and X-ray crystallography are revealing the structure of this important molecular machine. Both approaches have contributed to our understanding of the organization of the transmembrane helices of higher plant and cyanobacterial PS II and both indicate that PS II normally functions as a dimer. However the high-resolution electron density maps derived from X-ray crystallography currently at 3.7/3.8 A, have allowed assignments to be made to the redox active cofactors involved in the light-driven water-plastoquinone oxidoreductase activity and to the chlorophyll molecules that absorb and transfer energy to the reaction centre. In particular the X-ray work has identified density that can accommodate the four manganese atoms which catalyse the water-oxidation process. The Mn cluster is located at the lumenal surface of the DI protein and approximately 7 A from the redox active tyrosine residue (YZ) which acts an electron/proton transfer link to the primary oxidant P680.+. The lower resolution electron microscopy studies, however, are providing structural models of larger PS II supercomplexes that are ideal frameworks in which to incorporate the X-ray derived structures.