Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

Chloroplast-encoded small subunits of the three multiprotein complexes in photosynthetic electron transport

The photosystem I, photosystem II, and cytochromeb ₆ f complexes that are involved in electron transport of oxygenic photosynthesis consist of a number of subunits encoded by either the chloroplast or nuclear genomes. In addition to the major subunits that carry redox components or photosynthetic pigments, these complexes contain several to more than ten subunits with molecular masses of less than 10 kDa. Directed mutagenesis has served as a powerful tool for investigation of the roles of these small subunits in the organization or function of the complexes. Various chloroplast transformants of the green algaChlamydomonas reinhardtii and mutants of cyanobacteria in which a gene encoding a small subunit was deleted or altered have been constructed. Evidence has accumulated suggesting that these small subunits function in the assembly, stabilization, or protection from photoinhibition of the complexes or in the modulation or regulation of electron transport. This article presents an overview of the properties and functions of the chloroplast-encoded small subunits of the three multiprotein complexes of photosynthetic electron transport that have been mainly analyzed with chloroplast transformants ofC. reinhardtii and the corresponding cyanobacterial transformants. Recipient of the Botanical Society Award for Young Scientists, 1995.     

Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc 1 complex inSaccharomyces cerevisiae

The iron-sulfur protein of the cytochromebc ₁ complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochromec ₁ and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochromeb-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochromebc ₁ complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochromec ₁ and generates a strongly reducing ubisemiquinone anion radical which is proximal to theb-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.     

A proposed pathway of proton translocation through thebc complexes of mitochondria and chloroplasts

The cytochromebc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50–70% with minimal effect on electron transfer in the cytochromebc ₁ and cytochromebf complexes reconstituted into liposomes. Subsequent binding studies with cytochromebc ₁ and cytochromebf complexes indicate that DCCD specifically binds to the subunitb and subunitb ₆, respectively, in a time and concentration dependent manner. Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochromeb and aspartate 155 or glutamate 166 of spinach cytochromeb ₆. Moreover, similar inhibition of proton translocating activity and binding to cytochromeb and cytochromeb ₆ were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD. The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochromeb and cytochromeb ₆ is localized near the surface of the membrane but shielded from the external medium.     

Structural aspects of the cytochromeb 6 f complex; structure of the lumen-side domain of cytochromef

The following findings concerning the structure of the cytochromeb ₆ f complex and its component polypeptides, cytb ₆, subunit IV and cytochromef subunit are discussed:

Cytochrome b 6 f complex: Dynamic molecular organization,function and acclimation

The cytochrome b ₆ f complex occupies a central position in photosynthetic electron transport and proton translocation by linking PS II to PS I in linear electron flow from water to NADP⁺, and around PS I for cyclic electron flow. Cytochrome b ₆ f complexes are uniquely located in three membrane domains: the appressed granal membranes, the non-appressed stroma thylakoids and end grana membranes, and also the non-appressed grana margins, in contrast to the marked lateral heterogeneity of the localization of all other thylakoid multiprotein complexes. In addition to its vital role in vectorial electron transfer and proton translocation across the membrane, cytochrome b ₆ f complex is also involved in the regulation of balanced light excitation energy distribution between the photosystems, since its redox state governs the activation of LHC II kinase (the kinase that phosphorylates the mobile peripheral fraction of the chlorophyll a/b-proteins of LHC II of PS II). Hence, cytochrome b ₆ f complex is the molecular link in the interactive co-regulation of light-harvesting and electron transfer.The importance of a highly dynamic, yet flexible organization of the thylakoid membranes of plants and green algae has been highlighted by the exciting discovery that a lateral reorganization of some cytochrome b ₆ f complexes occurs in the state transition mechanism both in vivo and in vitro (Vallon et al. 1991). The lateral redistribution of phosphorylated LHC II from stacked granal membrane regions is accompanied by a concomitant movement of some cytochrome b ₆ f complexes from the granal membranes out to the PS I-containing stroma thylakoids. Thus, the dynamic movement of cytochrome b ₆ f complex as a multiprotein complex is a molecular mechanism for short-term adaptation to changing light conditions. With the concept of different membrane domains for linear and cyclic electron flow gaining credence, it is thought that linear electron flow occurs in the granal compartments and cyclic electron flow is localised in the stroma thylakoids at non-limiting irradiances. It is postulated that dynamic lateral reversible redistribution of some cytochrome b ₆ f complexes are part of the molecular mechanism involved in the regulation of linear electron transfer (ATP and NADPH) and cyclic electron flow (ATP only). Finally, the molecular significance of the marked regulation of cytochrome b ₆ f complexes for long-term regulation and optimization of photosynthetic function under varying environmental conditions, particularly light acclimation, is discussed.Abbreviations Chl chlorophyll - cyt cytochrome - PS Photosystem     

Interaction between cadmium, zinc and silver-substituted plastocyanin and cytochrome b 6 f complex — heavy metals toxicity towards photosynthetic apparatus

This paper reports the results of research on the interaction between the cytochrome f of the active cytochrome b ₆ f complex (incubated with Cd-, Zn-, and Ag-substituted plastocyanins) and Cu-plastocyanin. The presented studies show, that the metal derivatives of plastocyanin can have an influence on the photosynthetic electron transfer path: cytochrome b ₆ f complex — photosystem I. The metal-substituted plastocyanins occupy the plastocyanin electron transfer site of the cytochrome f. The stopped-flow measurements show, that although the metal derivatives of plastocyanin do not influence the rate of cyt f- Pc electron transfer, creation of the non-electron-transfer complexes characterised by a strong binding between the cyt f and substituted plastocyanins and their slow release, dependent on the redox state of the substituted metal, results in the decrease of a turnover of the cytochrome complex. The research was done in the Department of Plant Biochemistry, Freiburg University, Sch?nzlestrasse 1, 79 104 Freiburg, Germany     

Characterization of the photosynthetic electron transport chain in normal and photobleachedAnabaena cylindrica by flash spectroscopy

Electron transport of normal and photobleachedAnabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (540 and 566 nm) and two negative bands (515 and 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochromef and the reduction of cytochromeb ₅₆₃, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) sensitivity of cytochromef in normal cells, while DCMU was completely ineffective for cytochromef reduction in photobleached cells. The absorbance change of cytochromeb ₅₆₃ increased, while the absorbance change of cytochromef was smaller than in normal cells. The increased O₂ evolution in photobleached cells and the negligible electron transport via cytochromef suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleachedAnabaena cylindrica.     

Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b6f complex in maize

Nuclear genes essential for the biogenesis of the chloroplast cytochrome b ₆ f complex were identified by mutations that cause the specific loss of the complex. We describe four transposon-induced maize mutants that lack cytochrome b ₆ f proteins but contain normal levels of other photosynthetic complexes. The four mutations define two nuclear genes. To identify the step at which each mutation blocks protein accumulation, mRNAs encoding each subunit were examined by Northern hybridization analysis and the rates of subunit synthesis were examined in pulse-labeling experiments. In each mutant the mRNAs encoding the known subunits of the complex were normal in size and abundance and the major subunits were synthesized at normal rates. Thus, these mutations block the biogenesis of the cytochrome b ₆ f complex at a post-translational step. The two nuclear genes identified by these mutations may encode previously unknown subunits, be involved in prosthetic group synthesis or attachment, or facilitate assembly of the complex. These mutations were also used to provide evidence for the authenticity of a proposed fifth subunit of the complex and to demonstrate a role for the cytochrome b ₆ f complex in protecting photosystem 11 from light-induced degradation.     

Experimental and theoretical analysis of the interaction between cytochromec and cytochromeb 5

Experimental and theoretical investigation of the interaction of cytochromec and cytochromeb ₅ performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochromec and cytochromeb ₅ is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.     

Amino acid identities in the three redox center-carrying polypeptides of cytochromebc 1/b 6 f complexes

The comparison of primary structures is extended to 22 cytochromesb orb ₆, 12 cytochromesc ₁ orf, and 8 Rieske FeS proteins. Conclusions are drawn as to their phylogenetic relationship as well as on conserved, functionally important amino acids and secondary structures. The results are in favor of two independent quinone binding sites at opposite surfaces of the membrane, topping one of the two hemes of cytochromeb each.     

The cytochromec reductase/oxidase respiratory pathway ofParacoccus denitrificans: Genetic and functional studies

Data are presented on three components of the quinol oxidation branch of theParacoccus respiratory chain: cytochromec reductase, cytochromec ₅₅₂, and thea-type terminal oxidase. Deletion mutants in thebc ₁ and theaa ₃ complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochromec ₅₅₂ in electron transport between complex III and IV is presented.     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

Enhanced leaf photosynthesis as a target to increase grain yield: insights from transgenic rice lines with variable Rieske FeS protein content in the cytochrome b6/f complex

Although photosynthesis is the most important source for biomass and grain yield, a lack of correlation between photosynthesis and plant yield among different genotypes of various crop species has been frequently observed. Such observations contribute to the ongoing debate whether enhancing leaf photosynthesis can improve yield potential. Here, transgenic rice plants that contain variable amounts of the Rieske FeS protein _in the cytochrome (cyt) b₆/f complex between 10 and 100% of wild‐type levels have been used to investigate the effect of reductions of these proteins on photosynthesis, plant growth and yield. Reductions of the cyt b₆/f complex did not affect the electron transport rates through photosystem I but decreased electron transport rates through photosystem II, leading to concomitant decreases in CO₂ assimilation rates. There was a strong control of plant growth and grain yield by the rate of leaf photosynthesis, leading to the conclusion that enhancing photosynthesis at the single‐leaf level would be a useful target for improving crop productivity and yield both via conventional breeding and biotechnology. The data here also suggest that changing photosynthetic electron transport rates via manipulation of the cyt b₆/f complex could be a potential target for enhancing photosynthetic capacity in higher plants.     

Organization and function of cytochromeb and ubiquinone in the cristae membrane of beef heart mitochondria

The arrangement and function of the redox centers of the mammalianbc ₁ complex is described on the basis of structural data derived from amino acid sequence studies and secondary structure predictions and on the basis of functional studies (i.e., EPR data, inhibitor studies, and kinetic experiments). Two ubiquinone reaction centers do exist—a QH₂ oxidation center situated at the outer, cytosolic surface of the cristae membrane (Q₀ center), and a Q reduction center (Q _i center) situated more to the inner surface of the cristae membrane. The Q₀ center is formed by theb-566 domain of cytochromeb, the FeS protein, and maybe an additional small subunit, whereas the Q _i center is formed by theb-562 domain of cytochromeb and presumably the 13.4kDa protein (QP-C). The Q binding proteins are proposed to be protein subunits of the Q reaction centers of various multiprotein complexes. The path of electron flow branches at the Q₀ center, half of the electrons flowing via the high-potential cytochrome chain to oxygen and half of the electrons cycling back into the Q pool via the cytochromeb path connecting the two Q reaction centers. During oxidation of QH₂, 2H⁺ are released to the cytosolic space and during reduction of Q, 2H⁺ are taken up from the matrix side, resulting in a net transport across the membrane of 2H⁺ per e^– flown from QH₂ to cytochromec, the H⁺ being transported across the membrane as H (H⁺ + e^–) by the mobile carrier Q. The authors correct their earlier view of cytochromeb functioning as a H⁺ pump, proposing that the redox-linkedpK changes of the acidic groups of cytochromeb are involved in the protonation/deprotonation processes taking place during the reduction and oxidation of Q. The reviewers stress that cytochromeb is in equilibrium with the Q pool via the Q _i center, but not via the Q₀ center. Their view of the mechanisms taking place at the reductase is a Q cycle linked to a Q-pool where cytochromeb is acting as an electron pump.     

The chloroplast cytochrome b 6 f complex can exist in monomeric and dimeric states

The cytochrome b ₆ f complex isolated from spinach chloroplast membranes can be resolved into two forms, a monomeric and a dimeric form, by centrifugation on sucrose gradients. The conversion of the dimeric form of the complex into the monomeric form could be prevented by cross-linking with the homobifunctional reagent, dithiobis(succinimidylpropionate) but not by cross-linking with disuccinimidyltartrate or glutaraldehyde. SDS-PAGE analyses of the monomeric and dimeric forms of the cytochrome complex showed the presence of specific cross-linked products in each respective form of the complex. For example, the monomeric form contained a cross-linked product of cytochrome f, cytochrome b ₆ f and subunit IV while the dimeric form contained a cross-linked dimer of cytochrome b ₆ f. The presence of the former in the isolated cytochrome b ₆ f complex prepared by the method of Hurt and Hauska (Eur J Biochem 117: 591–599, 1981) indicates the presence of the monomer in his preparation.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DSP dithiobis(succinimidylpropionate) - DST disuccinimidyltartrate     

Glycinebetaine Stabilizes Photosystem 1 and Photosystem 2 Electron Transport in Spinach Thylakoid Membranes Against Heat Inactivation

Glycinebetaine, a compatible osmolyte of halotolerant plants and bacteria, partially protected photosystem (PS) 1 and PS2 electron transport reactions against thermal inactivation but with different efficiencies. In its presence, the temperature for half-maximal inactivation (t_1/2) was generally shifted downward by 3–12 °C. Glycinebetaine stabilized photoinduced oxygen evolving reactions of PS2 by protecting the tetranuclear Mn cluster and the extrinsic proteins of this complex. A weaker, although noticeable, stabilizing effect was observed in photoinduced PS2 electron transport reactions that did not originate in the oxygen-evolving complex (OEC). This weaker protection by glycinebetaine was probably exerted on the PS2 reaction centre. Glycinebetaine protected also photoinduced electron transport across PS1 against thermal inactivation. The protective effect was exerted on plastocyanin, the mobile protein in the lumen that carries electrons from the integral cytochrome b ₆ f complex to the PS1 complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

Thebc 1 complexes ofRhodobacter sphaeroides andRhodobacter capsulatus

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochromec oxidoreductase (bc ₁ complex). In bothRhodobacter sphaeroides andRhodobacter capsulatus, thebc ₁ complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because thebc ₁ complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochromeb subunit, in the Rieske iron-sulfur subunit, and in the cytochromec ₁ subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Q_o) site or the quinol reductase (Q_i) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.