Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

Direct monitoring of the electron pool effect of cytochrome<Emphasis Type="Italic"> c</Emphasis><Subscript>3</Subscript> by highly sensitive EQCM measurements

Cytochrome c₃ from Desulfovibrio vulgaris has four hemes per molecule, and a redox change at the hemes alters the conformation of the protein, leading to a redox-dependent change in the interaction of cytochrome c₃ with redox partners (an electron acceptor or an electron donor). The redox-dependent change in this interaction was directly monitored by the high-performance electrochemical quartz crystal microbalance (EQCM) technique that has been improved to give high sensitivity in solution. In this method, cytochrome c₃ molecules in solution associate electrostatically with a viologen-immobilized quartz crystal electrode as a monolayer, and redox of the associating cytochrome c₃ is controlled by the immobilized viologen. This technique makes it possible to measure the access of cytochrome c₃ to the electrode or repulsion from the electrode, and hence interconversion between an electrostatic complex and an electron transfer complex on the cytochrome c₃ and the viologen as a mass change accompanying a potential sweep is monitored. In addition, simultaneous measurement of a mass change and a potential step reveals that the cytochrome c₃ stores electrons when the four hemes are reduced (an electron pool effect), that is, the oxidized cytochrome c₃ facilitates acceptance of electrons from the immobilized viologen molecule, but the reduced cytochrome c₃ donates the accepted electrons to the viologen with difficulty.     

Structural basis for the mechanism of electron bifurcation at the quinol oxidation site of the cytochrome <Emphasis Type="Italic">bc</Emphasis><Subscript>1</Subscript> complex

At the heart of the Q cycle hypothesis, the cytochrome bc1 complex (bc1) is required to separate the two electrons from a quinol molecule at the quinol oxidation site. Recent studies have brought to light an intricate mechanism for this bifurcated electron transfer. A survey of the protein data bank shows 30 entries for the structures of bc1 and the homologous b6 f complex. These structures provide considerable insights into the structural organization of mitochondrial, bacterial, and plant enzymes. Crystallographic binding studies of bc1 with either quinone reduction (QN) and/or quinol oxidation (QP) site inhibitors offer atomic details on how these compounds interact with residues at their respective sites. Most importantly, the different locations and apparent flexibility observed in crystals for the extrinsic domain of the iron-sulfur protein (ISP) subunit suggest a mechanism for electron bifurcation at the QP site. Analyses of various inhibitor-bound structures revealed two classes of QP site inhibitors: Pm inhibitors that promote ISP mobility and Pf inhibitors that favor the fixation of the ISP conformation. Those analyses also shed light on a possible process by which the ISP motion switch is controlled. The first phase reduction of ISP is shown to be comparable to the reduction of the bL heme by pre-steady state kinetic analysis, whereas the second phase reduction of ISP share similar kinetics with the reduction of the bH heme. The reduction of cyt c1 is measured much slower, indicating that the reduced ISP remains bound at the QP site until the reduced heme bL is oxidized by the heme bH and supporting the existence of a control mechanism for the ISP motion switch.     

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.     

Variations of vinblastine accumulation and redox state affected by exogenous H<Subscript>2</Subscript>O<Subscript>2</Subscript> in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don

Effects of exogenous H₂O₂ application on vinblastine (VBL) and its precursors, vindoline (VIN), catharanthine (CAT) and α-3′,4′-anhydrovinblastine (AVBL), were measured in Catharanthus roseus seedlings in order to explore possible correlation of VBL formation with oxidative stress. VBL accumulation has previously been shown to be regulated by an in vitro H₂O₂-dependent peroxidase (POD)-like synthase. Experimental exposure of plants to different concentrations of H₂O₂ showed that endogenous H₂O₂ and alkaloid concentrations in leaves were positively elevated. The time-course variations of alkaloid concentrations and redox state, reflected by the concentrations of H₂O₂, ascorbic acid (AA), oxidative product of glutathione (GSSG) and POD activity, were significantly altered due to H₂O₂ application. The further correlation analysis between alkaloids and redox status indicated that VBL production was tightly correlated with redox status. These results provide a new link between VBL metabolisms and redox state in C. roseus.     

NMR-validated structural model for oxidized<Emphasis Type="Italic"> Rhodopseudomonas palustris</Emphasis> cytochrome<Emphasis Type="Italic"> c</Emphasis><Subscript>556</Subscript>

The structure of oxidized Rhodopseudomonas palustris cytochrome c ₅₅₆ has been modeled after that of high-spin cytochrome c from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes. The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin. In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals. The aim was to exploit the special restraints arising from the paramagnetism of the metal ion. A total of 43 residual-dipolar-coupling and 74 pseudocontact-shift restraints, which together sampled all regions of the protein, were used in conjunction with over 40 routinely obtained NOE distance restraints. A calculation procedure was undertaken combining the program MODELLER and the solution structure determination program PARAMAGNETIC DYANA, which includes paramagnetism-based restraints. The directions and magnitude of the magnetic susceptibility anisotropy tensor were also calculated. The approach readily provides useful results, especially for paramagnetic metalloproteins of moderate to large dimensions.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0511-2     

Cyanobacterial Photosystem I lacks specificity in its interaction with cytochrome c<Subscript>6</Subscript> electron donors

In cyanobacteria, plastocyanin and cytochrome c ₆, the alternate donor proteins to Photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I varies accordingly. In order to elucidate whether these changes in the electron donors’ properties correlate with complementary changes in the docking site of the corresponding photosystem, we have investigated the kinetics of reactions between three cytochrome c ₆ with isoelectric points of 5.6, 7.0 and 9.0, with Photosystem I particles from the same three genera of cyanobacteria which provided the cytochromes. The model systems compared here thus sample the full range of charge properties observed in cytochromes c ₆: acidic, basic and neutral. The rate constants and dependence on ionic strength for photosystem I reduction were distinctive for each cytochrome c ₆, but independent of Photosystem I. We conclude that the specific structural features of each cytochrome c ₆ dictate their different kinetic behaviours, whereas the three photosystems are relatively indiscriminate in docking with the electron donors.     

Computational insights into the O<Subscript>2</Subscript>-evolving complex of photosystem II

Mechanistic investigations of the water-splitting reaction of the oxygen-evolving complex (OEC) of photosystem II (PSII) are fundamentally informed by structural studies. Many physical techniques have provided important insights into the OEC structure and function, including X-ray diffraction (XRD) and extended X-ray absorption fine structure (EXAFS) spectroscopy as well as mass spectrometry (MS), electron paramagnetic resonance (EPR) spectroscopy, and Fourier transform infrared spectroscopy applied in conjunction with mutagenesis studies. However, experimental studies have yet to yield consensus as to the exact configuration of the catalytic metal cluster and its ligation scheme. Computational modeling studies, including density functional (DFT) theory combined with quantum mechanics/molecular mechanics (QM/MM) hybrid methods for explicitly including the influence of the surrounding protein, have proposed chemically satisfactory models of the fully ligated OEC within PSII that are maximally consistent with experimental results. The inorganic core of these models is similar to the crystallographic model upon which they were based, but comprises important modifications due to structural refinement, hydration, and proteinaceous ligation which improve agreement with a wide range of experimental data. The computational models are useful for rationalizing spectroscopic and crystallographic results and for building a complete structure-based mechanism of water-splitting in PSII as described by the intermediate oxidation states of the OEC. This review summarizes these recent advances in QM/MM modeling of PSII within the context of recent experimental studies.     

The semiquinone cycle. A hypothesis of electron transfer and proton translocation in cytochromebc-type complexes

The Q cycle and theb cycle are the main current models of action of the cytochromebc-type complexes of mitochondria, bacteria, and chloroplasts. Both are based on the concept, proposed in 1972, of two sequential one-electron oxidations of (ubi)quinol along two discrete pathways which operate at different redox potentials, and with bound semiubiquinone as an intermediate. The models differ in two respects, viz. in the pathway of electron transfer and the principle of linkage of electron transfer to proton translocation. In this article we outline a new model, called the semiquinone or, simply, SQ cycle, which is based on the electron transfer principles of theb cycle but which incorporates the Q cycle concept of direct coupling between electron transfer and proton translocation through action of ubiquinone.This paper is dedicated to the memory of Bob Casey, who died in Helsinki on the 2nd of August 1985.     

The influence of Sc doping on structural,electronic and optical properties of Be<Subscript>12</Subscript>O<Subscript>12</Subscript>, Mg<Subscript>12</Subscript>O<Subscript>12</Subscript> and Ca<Subscript>12</Subscript>O<Subscript>12</Subscript> nanocages: a DFT study

Density functional theory (DFT) calculations were used to study the effect of scandium doping on the structural, energetic, electronic, linear and nonlinear optical (NLO) properties of Be₁₂O₁₂, Mg₁₂O₁₂ and Ca₁₂O₁₂ nanoclusters. Scandium (Sc) doping on nanoclusters leads to narrowing of their E _g, which enhances their conductance greatly. Also, the polarizability (α) and first hyperpolarizability (β₀) of nanoclusters were dramatically increased as Be, Mg or Ca atoms are substituted with a Sc atom. Among all clusters, α and β₀ values for Sc-doped Ca₁₂O₁₂ were the largest. Consequently, the effect of the doping atom, as well as of cluster size, on electronic and optical properties was explored. Time dependent (TD)-DFT calculations were also carried out to confirm the β₀ values; the results show that the higher value of first hyperpolarizability belongs to Sc-doped Ca₁₂O₁₂, which has the smallest transition energy (ΔEgn). The results obtained show that these clusters can be candidates for using in electronic devices and NLO materials in industry.     

Promotion of Energy Transfer and Oxygen Evolution in Spinach Photosystem II by Nano-anatase TiO<Subscript>2</Subscript>

Being a proven photocatalyst, nano-anatase is capable of undergoing electron transfer reactions under light. In previous studies we had proven that nano-anatase improved photosynthesis and greatly promoted spinach growth. The mechanisms by which nano-anatase promotes energy transfer and the conversion efficiency of the process are still not clearly understood. In the present paper, we report the results obtained with the photosystem II (PSII) isolated from spinach and treated by nano-anatase TiO₂ and studied the effect of nano-anatase TiO₂ on energy transfer in PSII by spectroscopy and on oxygen evolution. The results showed that nano-anatase TiO₂ treatment at a suitable concentration could significantly change PSII microenvironment and increase absorbance for visible light, improve energy transfer among amino acids within PSII protein complex, and accelerate energy transport from tyrosine residue to chlorophyll a. The photochemical activity of PSII (fluorescence quantum yield) and its oxygen-evolving rate were enhanced by nano-anatase TiO₂. This is viewed as evidence that nano-anatase TiO₂ can promote energy transfer and oxygen evolution in PSII of spinach.     

Architecture of complex I and its implications for electron transfer and proton pumping

Proton pumping NADH:ubiquinone oxidoreductase (complex I) is the largest and remains by far the least understood enzyme complex of the respiratory chain. It consists of a peripheral arm harbouring all known redox active prosthetic groups and a membrane arm with a yet unknown number of proton translocation sites. The ubiquinone reduction site close to iron-sulfur cluster N2 at the interface of the 49-kDa and PSST subunits has been mapped by extensive site directed mutagenesis. Independent lines of evidence identified electron transfer events during reduction of ubiquinone to be associated with the potential drop that generates the full driving force for proton translocation with a 4H⁺/2e⁻ stoichiometry. Electron microscopic analysis of immuno-labelled native enzyme and of a subcomplex lacking the electron input module indicated a distance of 35-60 Å of cluster N2 to the membrane surface. Resolution of the membrane arm into subcomplexes showed that even the distal part harbours subunits that are prime candidates to participate in proton translocation because they are homologous to sodium/proton antiporters and contain conserved charged residues in predicted transmembrane helices. The mechanism of redox linked proton translocation by complex I is largely unknown but has to include steps where energy is transmitted over extremely long distances. In this review we compile the available structural information on complex I and discuss implications for complex I function.     

Kinetics of the electron transfer reaction of Cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>552</Subscript> adsorbed on biomimetic electrode studied by time-resolved surface-enhanced resonance Raman spectroscopy and electrochemistry

Cytochrome c ₅₅₂ (Cyt-c ₅₅₂) and its redox partner ba ₃ -oxidase from Thermus thermophilus possess structural differences compared with Horse heart cytochrome c (cyt-c)/cytochrome c oxidase (CcO) system, where the recognition between partners and the electron transfer (ET) process is initiated via electrostatic interactions. We demonstrated in a previous study by surface-enhanced resonance Raman (SERR) spectroscopy that roughened silver electrodes coated with uncharged mixed self-assembled monolayers HS–(CH₂) _n –CH₃/HS–(CH₂) _{n + 1}–OH 50/50, n = 5, 10 or 15, was a good model to mimic the Cyt-c ₅₅₂ redox partner. All the adsorbed molecules are well oriented on such biomimetic electrodes and transfer one electron during the redox process. The present work focuses on the kinetic part of the heterogeneous ET process of Cyt-c ₅₅₂ adsorbed onto electrodes coated with such mixed SAMs of different alkyl chain length. For that purpose, two complementary methods were combined. Firstly cyclic voltammetry shows that the ET between the adsorbed Cyt-c ₅₅₂ and the biomimetic electrode is direct and reversible. Furthermore, it allows the estimation of both the density surface coverage of adsorbed Cyt-c ₅₅₂ and the kinetic constants values. Secondly, time-resolved SERR (TR-SERR) spectroscopy showed that the ET process occurs without conformational change of the Cyt-c ₅₅₂ heme group and allows the determination of kinetic constants. Results show that the kinetic constant values obtained by TR-SERR spectroscopy could be compared to those obtained from cyclic voltammetry. They are estimated at 200, 150 and 40 s⁻¹ for the ET of Cyt-c ₅₅₂ adsorbed onto electrodes coated with mixed SAMs HS–(CH₂) _n –CH₃/HS–(CH₂) _{n + 1}–OH 50/50, n = 5, 10 or 15, respectively. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Determination of reduction potential of an engineered Cu<Subscript>A</Subscript> azurin by cyclic voltammetry and spectrochemical titrations

The reduction potentials of an engineered Cu_A azurin in its native and thermally denatured states have been determined using cyclic voltammetry and spectrochemical titrations. Using a 4,4-dipyridyl disulfide modified gold electrode, the reduction potentials of native and thermally denatured Cu_A azurin are the same within the experimental error (422±5 and 425±5 mV vs. NHE, respectively, in 50 mM ammonium acetate buffer, pH 5.1, 300 mM NaCl, 25 °C), indicating that the potential is that of a nonnative state. In contrast, using a didodecyldimethylammonium bromide (DDAB) film-pyrolytic graphite edge (PGE) electrode, the reduction potentials of native and thermally denatured Cu_A azurin have been determined to be 271±7 mV (50 mM ammonium acetate buffer, pH 5.1, 4 °C) and 420±1 mV (50 mM ammonium acetate buffer, pH 5.1, 25 °C), respectively. Spectroscopic redox titration using [Ru(NH₃)₅Py]²⁺ resulted in a reduction potential (254±4 mV) (50 mM ammonium acetate buffer, pH 5.1, 4 °C) similar to the value obtained using the DDAB film-PGE electrochemical method. Complete reoxidation of [Ru(NH₃)₅Py]²⁺-reduced Cu_A azurin is also consistent with the conclusion that this spectrochemical titration method using [Ru(NH₃)₅Py]²⁺ measures the reduction potential of native Cu_A azurin.Abbreviations CcO cytochrome c oxidase - N₂OR nitrous oxide reductase - ET electron transfer - CV cyclic voltammetry - NHE normal hydrogen electrode - DDAB didodecyldimethylammonium bromide - PGE pyrolytic graphite edge     

Microbial transformation of ginsenoside Rb<Subscript>1</Subscript> by <Emphasis Type="Italic">Acremonium strictum</Emphasis>

Preparative-scale fermentation of ginsenoside Rb₁ (1) with Acremonium strictum AS 3.2058 gave three new compounds, 12β-hydroxydammar-3-one-20 (S)-O-β-d-glucopyranoside (7), 12β, 25-dihydroxydammar-(E)-20(22)-ene-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (8), and 12β, 20 (R), 25-trihydroxydammar-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside (9), along with five known compounds, ginsenoside Rd (2), gypenoside XVII (3), ginsenoside Rg₃ (4), ginsenoside F₂ (5), and compound K (6). The structural elucidation of these metabolites was based primarily on one- and two-dimensional nuclear magnetic resonance and high-resolution electron spray ionization mass spectra analyses. Among these compounds, 2–6 are also the metabolites of ginsenoside Rb₁ in mammals. This result demonstrated that microbial culture parallels mammalian metabolism; therefore, A. strictum might be a useful tool for generating mammalian metabolites of related analogs of ginsenosides for complete structural identification and for further use in pharmaceutical research in this series of compounds. In addition, the biotransformation kinetics was also investigated.     

Redox tuning of the catalytic activity of soluble fumarate reductases from Shewanella

Many enzymes involved in bioenergetic processes contain chains of redox centers that link the protein surface, where interaction with electron donors or acceptors occurs, to a secluded catalytic site. In numerous cases these redox centers can transfer only single electrons even when they are associated to catalytic sites that perform two-electron chemistry. These chains provide no obvious contribution to enhance chemiosmotic energy conservation, and often have more redox centers than those necessary to hold sufficient electrons to sustain one catalytic turnover of the enzyme. To investigate the role of such a redox chain we analyzed the transient kinetics of fumarate reduction by two flavocytochromes c₃ of Shewanella species while these enzymes were being reduced by sodium dithionite. These soluble monomeric proteins contain a chain of four hemes that interact with a flavin adenine dinucleotide (FAD) catalytic center that performs the obligatory two electron–two proton reduction of fumarate to succinate. Our results enabled us to parse the kinetic contribution of each heme towards electron uptake and conduction to the catalytic center, and to determine that the rate of fumarate reduction is modulated by the redox stage of the enzyme, which is defined by the number of reduced centers. In both enzymes the catalytically most competent redox stages are those least prevalent in a quasi-stationary condition of turnover. Furthermore, the electron distribution among the redox centers during turnover suggested how these enzymes can play a role in the switch between respiration of solid and soluble terminal electron acceptors in the anaerobic bioenergetic metabolism of Shewanella.     

Protein profiling in F<Subscript>1</Subscript> and F<Subscript>2</Subscript> generations of two tomato genotypes differing in ripening time

Pericarp polypeptide profiles were analyzed at three ripening stages in the F₁ hybrid and the F₂ population from the cross between the accessions: LA1385 (Lycopersicon esculentum var. cerasiforme) and 804627 (L. esculentum, a homozygous genotype for the nor mutant). Six polymorphic polypeptides were observed in LA1385, while no polymorphic polypeptides among ripening stages was observed in 804627. On the other hand, some polypeptides in the F₁ hybrid were not observed in the parents whereas others were present in both parental genotypes and were unnoticeable in the hybrid genotype. From a cluster analysis on the protein profiles of the F₂ population, the differential expression of proteins allowed to distinguish mature green (MG) stage from the others two stages, while for breaker stage (BR) and red ripe stage, the genetic background was more important in forming groups. The differential expression of proteins could be associated with fruit morphology traits such as a 72 kDa polypeptide present in MG stage with fruit diameter, height and mass and a 47 kDa polypeptide found in BR with fruit shelf life.     

Expression, purification and characterization of the sulfite reductase hemo-subunit, SiR-HP, from Acidithiobacillus ferrooxidans

Sulfite reductase (SiR) is a large and soluble enzyme which catalyzes the transfer of six electrons from NADPH to sulfite to produce sulfide. The sulfite reductase flavoprotein (SiR-FP) contains both FAD and FMN, and the sulfite reductase hemoprotein (SiR-HP) contains an iron-sulfur cluster coupled to a siroheme. The enzyme is arranged so that the redox cofactors in the FAD-FMN-Fe(4)S(4)-Heme sequence make an electron pathway between NADPH and sulfite. Here we report the cloning, expression, and characterization of the SiR-HP of the sulfite reductase from Acidithiobacillus ferrooxidans. The purified SiR-HP contained a [Fe(4)S(4)] cluster. Site-directed mutagenesis results revealed that Cys427, Cys433, Cys472 and Cys476 were in ligating with the [Fe(4)S(4)] cluster of the protein.