NMR of Redox Proteins of Plants, Yeasts and Photosynthetic Bacteria

NMR spectroscopy has evolved dramatically over the past 15 years, establishing a new, reliable methodology for studying biomacromolecules at atomic resolution. The three-dimensional structure and dynamics of a biomolecule or a biomolecular complex is only one of the main types of information available using NMR. The spectral assignment to the specific nuclei of a biostructure is a very precise reflection of their electronic environment. Any change in this environment due to a structural change, the binding of a ligand or the redox state of a redox cofactor, will be very sensitively reported by changes in the different NMR parameters. The capabilities of the NMR method are currently expanding dramatically and it is turning into a powerful means to study biosystems dynamically in exchange between different conformations, exchanging ligands, transient complexes, or the activation/inhibition of regulated enzymes. We review here several NMR studies that have appeared during the past 5 or 6 years in the field of redox proteins of plants, yeasts and photosynthetic bacteria. These new results illustrate the recent biomolecular NMR evolution and provide new physiological models for understanding the different types of electron transfer, including glutaredoxins, thioredoxins and their dependent enzymes, the ferredoxin-NADP oxidoreductase complex, flavodoxins, the plastocyanin-cytochrome f complex, and cytochromes c.     

A Comprehensive Phylogenetic Analysis of Rieske and Rieske-Type Iron-Sulfur Proteins

The Rieske iron-sulfur center consists of a [2Fe-2S] cluster liganded to a protein via two histidine and two cysteine residues present in conserved sequences called Rieske motifs. Two protein families possessing Rieske centers have been defined. The Rieske proteins occur as subunits in the cytochrome bc1 and cytochrome b6f complexes of prokaryotes and eukaryotes or form components of archaeal electron transport systems. The Rieske-type proteins encompass a group of bacterial oxygenases and ferredoxins. Recent studies have uncovered several new proteins containing Rieske centers, including archaeal Rieske proteins, bacterial oxygenases, bacterial ferredoxins, and, intriguingly, eukaryotic Rieske oxygenases. Since all these proteins contain a Rieske motif, they probably form a superfamily with one common ancestor. Phylogenetic analyses have, however, been generally limited to similar sequences, providing little information about relationships within the whole group of these proteins. The aim of this work is, therefore, to construct a dendrogram including representatives from all Rieske and Rieske-type protein classes in order to gain insight into their evolutionary relationships and to further define the phylogenetic niches occupied by the recently discovered proteins mentioned above.     

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.     

A comparative study of the thermal stability of plastocyanin, cytochrome c6 and Photosystem I in thermophilic and mesophilic cyanobacteria

Cytochrome c₆ (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Controlling Action of Actithiazic Acid on the Biosynthesis of Biotin-vitamers by Various Microorganisms

By the addition of actithiazic acid, or acidomycin (ACM), to culture media, the accumulation of desthiobiotin by various microorganisms was enhanced from two-fold to about seventyfold, while that of biotin was markedly reduced. Especially, Bacillus sphaericus accumulated 350 μg per ml of biotin-vitamers assayed with Saccharomyces cerevisiae. ACM was not incorporated into the desthiobiotin molecule by resting cells of B. sphaericus. The amount of biotin-vitamers assayed with S. cerevisiae which was synthesized from pimelic acid by the resting cells grown with ACM was twice as great as that synthesized by the cells grown without ACM. From these results, the mechanism of the controlling action of ACM on biotin biosynthesis was discussed.     

On the role of cytochrome c8 in photosynthetic electron transfer of the purple non-sulfur bacterium Rhodoferax fermentans

We report on the isolation, purification and functional characterization of a soluble c-type cytochrome from light-grown cells of the purple phototroph Rhodoferax fermentans. This cytochrome is basic (pI = 8), has a molecular mass of 12 kDa, and is characterized by a midpoint reduction potential of +285 mV. Partial analysis of the N-terminus amino-acid sequence shows a high similarity with cytochromes of c₈ type (formerly called Pseudomonas cytochrome c-551 type). Time-resolved spectrophotometric studies show that this cytochrome c₈ reduces the tetraheme subunit of the photosynthetic reaction center, in a fast (sub-ms) and a slow (ms) phase. Competition experiments in the presence of both cytochrome c₈ and high potential iron-sulfur protein (HiPIP), isolated from the same microorganism, show that cytochrome c₈ oxidation is decreased upon addition of HiPIP. These observations suggest that cytochrome c₈ and HiPIP might play alternative roles in the photosynthetic electron flow of Rhodoferax fermentans.     

Negatively charged residues in the H loop of PsaB subunit in Photosystem I from Synechocystis sp. PCC 6803 appear to be responsible for electrostatic repulsions with plastocyanin*

Wild-type plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803 does not form any kinetically detectable transient complex with Photosystem I (PS I) during electron transfer, but the D44R/D47R double mutant of copper protein does [De la Cerda et al. (1997) Biochemistry 36: 10125–10130]. To identify the PS I component that is involved in the complex formation with the D44R/D47R plastocyanin, the kinetic efficiency of several PS I mutants, including a PsaF–PsaJ-less PS I and deletion mutants in the lumenal H and J loops of PsaB, were analyzed by laser flash absorption spectroscopy. The experimental data herein suggest that some of the negative charges at the H loop of PsaB are involved in electrostatic repulsions with mutant plastocyanin. Mutations in the J loop demonstrate that this region of PsaB is also critical. The interaction site of PS I is thus not as defined as first expected but much broader, thereby revealing how complex the evolution of intermolecular electron transfer mechanisms in photosynthesis has been. This revised version was published online in June 2006 with corrections to the Cover Date.     

Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D ¹H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D ¹H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R _2p of the resonances can be determined, and the conditions kR _2p or _pkR _2p are fulfilled, _p being the frequency separation between corresponding diamagnetic and paramagnetic sites.     

Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer

In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemes b. The hemes display a large difference in redox midpoint potentials (ΔE_{m_}b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on heme b ligand mutants of cytochrome bc₁ in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔE_{m_}b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔE_{m_}b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functional in vivo. This reveals that cytochrome bc₁ can accommodate large changes in ΔE_{m_}b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemes b in this cytochrome and in other membranous cytochromes b act as electronic connectors for the catalytic sites with no fine tuning in ΔE_{m_}b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential.     

Characterisation of cytochrome c6from Chlorella fusca

A c-type monohaem, cytochrome c₆was isolated from a soluble extract of the green alga Chlorella fusca. The isolated protein shows an apparent molecular mass of 10 kDa by SDS-PAGE, but behaves as a dimer of 20.3 kDa in gel-filtration; the isoelectric point is 3.6. The N-terminal sequence shows high identity with other green algae cytochromes c₆. The mid-point redox potential is about +350 mV between pH 5 and 9. The ferric and ferrous forms, and their pH equilibria, have been studied using visible, CD and EPR spectroscopies. The visible spectrum of the reduced cytochrome c₆is typical of a c-type haem protein, with maxima at 274 nm, 318 nm (-peak), 416 nm (-peak), 522 nm (-peak), 552–553 nm (-peak). A 690 nm band, characteristic of a haem Met-His axial coordination of the haem group, is present in the oxidized form. At high pH values ( 8), cytochrome c₆undergoes an alkaline transition, with a pKa of 8.7. Between pH 3 and 9 the EPR spectrum is dominated by two rhombic species, with g-values at 3.32, 2.05, 1.05 and 2.96, 2.30, 1.43, which interconvert with a pK_aof 4. CD spectrum of Chlorella fusca cytochrome c₆shows that the proteins must be mainly built up by -helices. Even though there are similarities between Chlorella fusca cytochrome c₆and that isolated from Monoraphidium braunii, no cross-reactivity with the antibodies raised against the Chlorella fusca cytochrome has been detected for the protein from Monoraphidium braunii.     

The cytochrome c folding landscape revealed by electron-transfer kinetics

We have investigated the folding energy landscape of cytochrome c by exploiting the widely different electron-transfer (ET) reactivities of buried and exposed Zn(II)-substituted hemes. An electronically excited Zn-porphyrin in guanidine hydrochloride denatured Zn-substituted cytochrome c (Zn-cyt c) reduces ruthenium(III) hexaammine about ten times faster than when embedded in the fully folded protein. Measurements of ET kinetics during Zn-cyt c folding reveal a burst intermediate in which one-third of the ensemble has a protected Zn-porphyrin and slow ET kinetics; the remaining fraction exhibits fast ET characteristic of a solvent-exposed redox cofactor. The ET data show that, under solvent conditions favoring the folded protein, collapsed non-native structures are not substantially more stable than extended conformations, and that the two populations interchange rapidly. Most of the folding free energy, then, is released when compact structures evolve into the native fold.     

A quick solution structure determination of the fully oxidized double mutant K9-10A cytochrome c7 from Desulfuromonas acetoxidans and mechanistic implications

Lysines 9 and 10 in Desulfuromonas acetoxidans cytochrome c₇, which could be involved in the interaction mechanism with the redox partners, have been replaced by alanine residues using site-directed mutagenesis. The solution structure of the fully oxidized form of K9-10A cytochrome c₇, which is paramagnetic with three paramagnetic centers, has been determined via ¹H NMR. The assignment of the spectra has been performed through an automatic program whose algorithm and strategy are here described. The assignment of the NOESY spectra has been further extended by back calculating the NOESY maps. The final number of meaningful NOE-based upper distance limits was 1186. In the Restrained Energy Minimization calculations, 147 pseudocontact shift constraints were also included, which showed consistency with NOE-based constraints and therefore further contribute to validate the structure quality. A final family of 35 conformers was calculated with RMSD values with respect to the mean structure of 0.69 ± 0.17 Å and 1.05 ± 0.14 Å for the backbone and heavy atoms, respectively. The overall fold of the molecule is maintained with respect to the native protein. The loop present between heme III and heme IV results to be highly disordered also in the present structure although its overall shape mainly resembles that of the oxidized native protein, and the two strands which give rise to the short -sheet present at the N-terminus and connected by a turn containing the mutated residues, are less clearly defined. If this loop is neglected, the RMSD values are 0.52 ± 0.07 Å and 0.92 ± 0.06 Å for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The relative distances and orientations of the three hemes are maintained, as well as the orientation of the imidazole rings of the axial histidine ligands, with the only exception of heme IV. Such difference probably reflects minor conformational changes due to the substitution of the vicinal Lys10 with an Ala. The replacement of the two lysines does not affect the reduction potentials of the three hemes, consistently with the expectations on the basis of the structure and electrostatic calculations. However, the replacement of the two lysines affects the reactivity of the mutant cytochrome c₇ with [Fe] hydrogenase, inducing a change in K _m. This finding is in agreement with the identification of the protein area around heme IV as the interacting site.     

Structural comparison of cytochrome c2and cytochrome c6

Photosynthesis Research - This review compares the three-dimensional structures of the solublec-type cytochromes that functionally link membrane-bound energy transducingcomplexes in algal,...     

Redox potentials of the blue copper sites of bilirubin oxidases

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was > 650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.     

The Cytochrome bc 1 Complex and its Homologue the b 6 f Complex: Similarities and Differences

The ubihydroquinone:cytochrome c oxidoreductase (also called complex III, or bc (1) complex), is a multi subunit enzyme encountered in a very broad variety of organisms including uni- and multi-cellular eukaryotes, plants (in their mitochondria) and bacteria. Most bacteria and mitochondria harbor various forms of the bc (1) complex, while plant and algal chloroplasts as well as cyanobacteria contain a homologous protein complex called plastohydroquinone:plastocyanin oxidoreductase or b (6) f complex. Together, these enzyme complexes constitute the superfamily of the bc complexes. Depending on the physiology of the organisms, they often play critical roles in respiratory and photosynthetic electron transfer events, and always contribute to the generation of the proton motive force subsequently used by the ATP synthase. Primarily, this review is focused on comparing the 'mitochondrial-type' bc (1) complex and the 'chloroplast-type' b (6) f complex both in terms of structure and function. Specifically, subunit composition, cofactor content and assembly, inhibitor sensitivity, proton pumping, concerted electron transfer and Fe-S subunit large-scale domain movement of these complexes are discussed. This is a timely undertaking in light of the structural information that is emerging for the b (6) f complex.     

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.     

Supramolecular organization of the photosynthetic chain in chromatophores and cells of Rhodobacter sphaeroides

Flash-induced kinetics of the membrane potential increase related to electron transfer within the cytochrome (cyt) b/c₁ complex (Phase III) and that of cyt c₁+c₂ reduction have been measured as a function of myxothiazol concentration in isolated chromatophores and whole cells of Rhodobacter sphaeroides. Upon addition of nonsaturating concentrations of myxothiazol, kinetics of Phase III display two phases, Phase IIIa and Phase IIIb. The amplitude of Phase IIIa, completed in about 10 ms, is proportional to the fraction of non-inhibited cyt b/c₁ complexes, while its half-time is independent of the myxothiazol concentration. A fast cyt c₁+c₂ reduction phase is correlated to Phase IIIa. These experiments demonstrate that, in a range of time of several ms, diffusion of cyt c₂ is restricted to domains formed by a supercomplex including two reaction centers (RCs) and a single cyt b/c₁ complex, as proposed by Joliot et al. (Biochim Biophys Acta 975: 336–345, 1989). Phase IIIb, completed in about 100 ms, shows that positive charges or inhibitor molecules are exchanged between supercomplexes in this range of time. These exchanges occur within domains including 2 to 3 supercomplexes, i.e. in membrane domains smaller than a single chromatophore. These conclusions apply to both isolated chromatophores and whole cells.Abbreviations cyt cytochrome - MOPS 3-(N-morpholino)propane sulfonic acid - PMS phenazine methosulfate - P primary donor - Rb. Rhodobacter - RC reaction center     

On an Inhibitor of Ferredoxin Catalyzed Cyclic Photophosphorylation in Thylakoid Membranes*

Dibromo- and diiodo-naphthoquinones are shown to be inhibitors of the cytochrome b₆/f complex in isolated thylakoid membranes from spinach chloroplasts. Dibromo-naphthoquinone inhibits ferredoxin catalyzed cyclic photophosphorylation at 0.1 μM concentrations, but non cyclic e-flow only at 10 μM. It does not inhibit cyclic systems with artifical cofactors, nor non-cyclic electron flow from duroquinol through photosystem I via the cytochrome b₆/f complex. Dibromo-naphthoquinone does however, lower the stoichiometry for ATP formation in the duroquinol donor system. This inhibitory pattern is quite different from that of DBMIB, but very similar to that of antimycin. This antimycin-like behaviour of these inhibitors is interpreted to indicate a) the existence of a Q_c site in the cytochrome b₆/f complex and its obligate function in ferredoxin catalyzed cyclic electron flow and b) a non-essential role of the Q_c site in non-cyclic electron flow, but which — when operative — pumps an extra proton across the thylakoid membrane increasing the ATP yield.