Molecular dynamics simulations give insight into the conformational change,complex formation,and electron transfer pathway for cytochrome P450 reductase

Cytochrome P450 reductase (CYPOR) undergoes a large conformational change to allow for an electron transfer to a redox partner to take place. After an internal electron transfer over its cofactors, it opens up to facilitate the interaction and electron transfer with a cytochrome P450. The open conformation appears difficult to crystallize. Therefore, a model of a human CYPOR in the open conformation was constructed to be able to investigate the stability and conformational change of this protein by means of molecular dynamics simulations. Since the role of the protein is to provide electrons to a redox partner, the interactions with cytochrome P450 2D6 (2D6) were investigated and a possible complex structure is suggested. Additionally, electron pathway calculations with a newly written program were performed to investigate which amino acids relay the electrons from the FMN cofactor of CYPOR to the HEME of 2D6. Several possible interacting amino acids in the complex, as well as a possible electron transfer pathway were identified and open the way for further investigation by site directed mutagenesis studies.     

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.     

导电材料强化微生物直接种间电子传递产甲烷的研究进展

厌氧条件下,微生物可以通过厌氧代谢产生甲烷(CH_4),由此衍生的厌氧消化技术可实现能源的回收利用。产CH_4的关键步骤是刺激发酵细菌和产甲烷古菌之间的有效电子转移,电活性微生物可以取代传统的氢/甲酸盐实现直接种间电子传递,其电子传递效率更高。添加导电材料可以促进直接种间电子传递并提高CH_4产率,是一种更有效的强化电子传递方式。本文在梳理直接种间电子传递发展和机理的基础上,综述了常见的促进直接种间电子传递的碳基和铁基导电材料,对其结构特征、电子传递机理、强化产CH_4和中间产物消耗等方面进行了系统总结。旨在为导电材料促进直接种间电子传递的研究提供参考,并探讨了未来可能的研究方向。     

Electron transfer between the quinones in the photosynthetic reaction center and its coupling to conformational changes

The electron transfer between the two quinones Q(A) and Q(B) in the bacterial photosynthetic reaction center (bRC) is coupled to a conformational rearrangement. Recently, the X-ray structures of the dark-adapted and light-exposed bRC from Rhodobacter sphaeroides were solved, and the conformational changes were characterized structurally. We computed the reaction free energy for the electron transfer from to Q(B) in the X-ray structures of the dark-adapted and light-exposed bRC from Rb. sphaeroides. The computation was done by applying an electrostatic model using the Poisson-Boltzmann equation and Monte Carlo sampling. We accounted for possible protonation changes of titratable groups upon electron transfer. According to our calculations, the reaction energy of the electron transfer from to Q(B) is +157 meV for the dark-adapted and -56 meV for the light-exposed X-ray structure; i.e., the electron transfer is energetically uphill for the dark-adapted structure and downhill for the light-exposed structure. A common interpretation of experimental results is that the electron transfer between and Q(B) is either gated or at least influenced by a conformational rearrangement: A conformation in which the electron transfer from to Q(B) is inactive, identified with the dark-adapted X-ray structure, changes into an electron-transfer active conformation, identified with the light-exposed X-ray structure. This interpretation agrees with our computational results if one assumes that the positive reaction energy for the dark-adapted X-ray structure effectively prevents the electron transfer. We found that the strongly coupled pair of titratable groups Glu-L212 and Asp-L213 binds about one proton in the dark-adapted X-ray structure, where the electron is mainly localized at Q(A), and about two protons in the light-exposed structure, where the electron is mainly localized at Q(B). This finding agrees with recent experimental and theoretical studies. We compare the present results for the bRC from Rb. sphaeroides to our recent studies on the bRC from Rhodopseudomonas viridis. We discuss possible mechanisms for the gated electron transfer from to Q(B) and relate them to theoretical and experimental results.     

Electron transfer facilitated by superoxide dismutase: a model for membrane redox systems?

Membranes, which are an amalgam of proteins and lipids, effect electron transfer through largely unknown mechanisms. Using albumin with bound fatty acids as a model, we have investigated the possible role of these two membrane constituents in electron transfer. In the presence of albumin: fatty acid, there is substantial enhancement of the reduction of ferricytochrome C by ferrous iron. To assess the possible role of free superoxide in cytochrome C reduction, we added mammalian copper/zinc containing superoxide dismutase (Cu/Zn SOD), which catalyzes the transfer of electrons between superoxide anion radicals, forming oxygen and hydrogen peroxide. Surprisingly, in the presence of either albumin or fatty acid free albumin, Cu/Zn SOD actually accelerates electron transfer from ferrous iron to ferricytochrome C. By contrast, neither inactive Cu/Zn SOD nor active manganese SOD facilitates the ferrous iron-dependent reduction of cytochrome C. These results suggest that, in some circumstances, Cu/Zn SOD may transfer electrons to alternative acceptors and that such transfer depends upon the unique reduction/oxidation reaction mechanism of Cu/Zn SOD. If so, this ubiquitous enzyme could be involved in regulating cellular electron transfer reactions as well as acting as a superoxide 'detoxify-ing' agent.     

Redox-linked proton translocation in cytochrome oxidase: the importance of gating electron flow. The effects of slip in a model transducer.

In at least one component of the mitochondrial respiratory chain, cytochrome c oxidase, exothermic electron transfer reactions are used to drive vectorial proton transport against an electrochemical hydrogen ion gradient across the mitochondrial inner membrane. The role of the gating of electrons (the regulation of the rates of electron transfer into and out of the proton transport site) in this coupling between electron transfer and proton pumping has been explored. The approach involves the solution of the steady-state rate equations pertinent to proton pump models which include, to various degrees, the uncoupled (i.e., not linked to proton pumping) electron transfer processes which are likely to occur in any real electron transfer-driven proton pump. This analysis furnishes a quantitative framework for examining the effects of variations in proton binding site pKas and metal center reduction potentials, the relationship between energy conservation efficiency and turnover rate, the conditions for maximum power output or minimum heat production, and required efficiency of the gating of electrons. Some novel conclusions emerge from the analysis, including: An efficient electron transfer-driven proton pump need not exhibit a pH-dependent reduction potential; Very efficient gating of electrons is required for efficient electron transfer driven proton pumping, especially when a reasonable correlation of electron transfer rate and electron transfer exoergonicity is assumed; and A consideration of the importance and possible mechanisms of the gating of electrons suggests that efficient proton pumping by CuA in cytochrome oxidase could, in principle, take place with structural changes confined to the immediate vicinity of the copper ion, while proton pumping by Fea would probably require conformational coupling between the iron and more remote structures in the enzyme. The conclusions are discussed with reference to proton pumping by cytochrome c oxidase, and some possible implications for oxidative phosphorylation are noted.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Electron transfer pathways in cytochrome c oxidase

Mixed quantum mechanical/molecular mechanics calculations were used to explore the electron pathway of the terminal electron transfer enzyme, cytochrome c oxidase. This enzyme catalyzes the reduction of molecular oxygen to water in a multiple step process. Density functional calculations on the three redox centers allowed for the characterization of the electron transfer mechanism, following the sequence Cu(A)→heme a→heme a(3). This process is largely affected by the presence of positive charges, confirming the possibility of a proton coupled electron transfer. An extensive mapping of all residues involved in the electron transfer, between the Cu(A) center (donor) and the O(2) reduction site heme a(3)-Cu(B) (receptor), was obtained by selectively activating/deactivating different quantum regions. The method employed, called QM/MM e-pathway, allowed the identification of key residues along the possible electron transfer paths, consistent with experimental data. In particular, the role of arginines 481 and 482 appears crucial in the Cu(A)→heme a and in the heme a→heme a(3) electron transfer processes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Attempts to observe through-chain energy transfer and electron transfer on α-helical polypeptide chain

Singlet singlet energy transfer between the two terminal chromophores attached to an α-helical polypeptide chain has been studied. The transfer efficiency was satisfactorily explained by Förster's theory when the interchromophore distance was calculated from the α-helical structure. Therefore, it was concluded that no particular effect from the possible energy band structure of the α-helical conformation was detected in the end-to-end energy transfer. Similarly, end-to-end electron transfer was attempted between the electron donor acceptor pair attached to the ends of α-helcial polypeptide chain. However, no intramolecular interaction was found between the donor acceptor pair, indicating that the exciton structure of the α-helical polypeptides is not effective enough to realize through-chain electron transfer.     

Blue light drives B-side electron transfer in bacterial photosynthetic reaction centers.

The core of the photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer. Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near-infrared light. Here, it is shown that excitation with blue light into the Soret band of the reaction center gives rise to electron transfer along the alternate or B-side pathway, resulting in a charge-separated state involving the anion of the B-side bacteriopheophytin. This electron transfer is much faster than normal A-side transfer, apparently occurring within a few hundred femtoseconds. At low temperatures, the B-side charge-separated state is stable for at least 1 ns, but at room temperature, the B-side bacteriopheophytin anion is short-lived, decaying within approximately 15 ps. One possible physiological role for B-side electron transfer is photoprotection, rapidly quenching higher excited states of the reaction center.     

NMR study of the interaction of plastocyanin with chromium(III) analogues of inorganic electron transfer reagents

High resolution nuclear magnetic resonance spectroscopy has been used to examine the interaction of plastocyanins from French bean (Phaseolus vulgaris) and cucumber (Cucumis sativus) with three complexes—potassium hexacyanochromate(III), hexamminechromium(III) nitrate and tris(1,10-phenanthroline)-chromium(III) perchlorate—which are analogues of inorganic electron transfer reagents. The results indicate a high degree of specificity in the binding of these complexes and two binding sites on the protein are identified. One binding site is situated close to the copper atom and is clearly suited to outer sphere electron transfer through one of the histidine ligands. The other binding site is more distant from the copper atom and this mechanism cannot be operative. Electron transfer via hydrophobic channels or electron tunneling are possible mechanisms of electron transfer.     

Kinetics of phyllosemiquinone oxidation in the Photosystem I reaction centre of Acaryochloris marina

Light-induced electron transfer reactions in the chlorophyll a/d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88±6ns and 345±10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5-30ns and 200-300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed.     

Interaction between enzyme-generated triplet carbonyls and molecules intercalated into DNA

The phosphorescence from enzyme-generated and -protected triplet acetone is very efficiently quenched by dyes intercalated into DNA. The process is unlikely to be due to energy transfer and is tentatively ascribed to electron transfer occurring within the DNA helix complex with the acting enzyme. This quenching markedly protects DNA from breaks induced by triplet acetone. In the case of some barely emissive enzyme-generated triplet carbonyl species, it is possible to detect a weak emission resulting from the interaction with dye X DNA; this emission may be associated with back electron transfer.     

Removal of PsaF alters forward electron transfer in photosystem I: evidence for fast reoxidation of QK-A in subunit deletion mutants of Synechococcus sp. PCC 7002

Recent studies of point mutations in photosystem I have suggested that the two kinetic phases of phylloquinone reoxidation represent electron transfer in the two branches of cofactors. This interpretation implies that changes in the relative amplitudes of the two kinetic phases represent a change in the extent of electron transfer in the two branches. Using time-resolved electron paramagnetic resonance (EPR), this issue is investigated in subunit deletion mutants of Synechococcus sp. PCC 7002. The spin-polarized EPR signals of P(700)(+)A(1)(-) and P(700)(+)FeS(-), both at room temperature and in frozen solution, are altered by deletion of PsaF and/or PsaE, and the differences from the wild type are much more pronounced in PS I complexes isolated from the mutants using Triton X-100 rather than n-dodecyl beta-d-maltopyranoside. The changes in the transient EPR data for the mutant complexes are consistent with a significant fraction of reaction centers showing (i) faster electron transfer from A(1)(-) to F(X), (ii) slower forward electron transfer from A(0)(-) to A(1), and (iii) slightly altered quinone hyperfine couplings, possibly as a result of a change in the hydrogen bonding. The fraction of fast electron transfer and its dependence on the isolation procedure are estimated approximately from simulations of the room temperature EPR data. The results are discussed in terms of possible models for the electron transfer. It is suggested that the detergent-induced fraction of fast electron transfer is most likely due to alteration of the environment of the quinone in the PsaA branch of cofactors and is not the result of a change in the directionality of electron transfer.     

The water channel of cytochrome c oxidase: inferences from inhibitor studies.

Cytochrome c oxidase couples electron transfer to proton transfer from inside the mitochondrion to the cytosol. Protons pass through a channel; it is closed except when protons are pumped. Electron transfer is also coupled to a water cycle. Water moves into and out of the oxidase during electron transfer, presumably through a channel. The three processes are coupled because of the common dependence on electron transfer. If water and protons had to pass through the same channel for the proton to pass, it might be possible to block the pore by entraining small molecules in the flow. The data in this report indicate that there is a correlation between the ability of a compound to inhibit the oxidase and its size. Formamide and formaldehyde are potent inhibitors. Larger and smaller molecules are poor inhibitors. Formamide introduces an internal block in electron transfer. It is a slow-onset, reversible inhibitor, dependent on turnover to manifest its effects. Vesicular oxidase is less influenced by formamide than is soluble oxidase; formamide must pass a permeability barrier to act. The data are consistent with a proton channel with constrictions at both ends that open to yield a pore of approximately 4 A.     

Substrate-mediated electron transfer in peptidylglycine alpha-hydroxylating monooxygenase.

Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 A apart on either side of a solvent-filled interdomain cleft, and the PHM reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. Our experiments show that PHM catalytic core (PHMcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the PHM catalytic cycle.     

Electron transfer in peptides and proteins

Proteins and peptides use their amino acids as medium for electron-transfer reactions that occur either in single-step superexchange or in multistep hopping processes. Whereas the rate of the single-step electron transfer dramatically decreases with the distance, a hopping process is less distance dependent. Electron hopping is possible if amino acids carry oxidizable side chains, like the phenol group in tyrosine. These side chains become intermediate charge carriers. Because of the weak distance dependency of hopping processes, fast electron transfer over very long distances occurs in multistep reactions, as in the enzyme ribonucleotide reductase.     

Are dietary carotenoids beneficial? Reactions of carotenoids with oxy-radicals and singlet oxygen.

Carotenoids play diverse roles in biology and medicine. Both the quenching of singlet oxygen (energy transfer) and interaction with oxy-radicals (electron transfer, H-atom transfer and addition reactions) are key processes in understanding many of these roles. Much previous work in 'simple' solvents is reviewed and new results in cell membrane models are presented. The possible consequences of using carotenoids as dietary supplements are discussed.     

Role of mediators in electron transport from glucose oxidase redox centre to electrode surface in a covalently coupled enzyme paste electrode

We have studied the glucose oxidase immobilized carbon paste electrodes in the presence and absence of small mediator molecules. We have used p-benzoquinone and riboflavin as mediators in our studies. The effect of mediator molecules on the electron transfer between the enzyme redox centre and the electrode surface was explained from the cyclic voltammograms and rotating disk electrode data. In the absence of oxygen, we have noted that the mediators play a central role in the electron transfer. We have also proposed a possible mechanism for the electron transfer from enzyme active site to the electrode surface via mediators, based on our observations. Dedicated to the memory of Professor J Das     

Concerning the enigmatic cytochrome b-559 of oxygenic photosynthesis

Photosynthesis Research - Although there is an extensive literature on the properties and possible electron transfer pathways of cytochrome b-559, which is a prominent subunit of the multi-subunit...