Cytochrome f: Structure,function and biosynthesis

Cytochrome f is an intrinsic membrane component of the cytochrome bf complex, transferring electrons from the Rieske FeS protein to plastocyanin in the thylakoid lumen. The protein is held in the thylakoid membrane by a single transmembrane span located near its C-terminus with a globular hydrophilic domain extending into the lumen. The globular domain of the turnip protein has recently been crystallised, offering the prospect of a detailed three-dimensional structure. Reaction with plastocyanin involves localised positive charges on cytochrome f interacting with the acidic patch on plastocyanin and electron transfer via the surface-exposed tyrosine residue (Tyr83) of plastocyanin. Apocytochrome f is encoded in the chloroplast genome and is synthesised with an N-terminal presequence which targets the protein to the thylakoid membrane. The synthesis of cytochrome f is coordinated with the synthesis of the other subunits of the cytochrome bf complex.     

Inhibitor binding to isolated chloroplast cytochrome bf complex

Effects of three inhibitors of quinol oxidation in the chloroplast cytochrome bf complex (stigmatellin, tridecylstigmatellin and dibromothymoquinone) were studied in an isolated system comprising Photosystem I (PS I) particles, plastocyanin (PC) and cytochrome bf complex, in the absence of quinol or quinone. Addition of these inhibitors increased the extent of cytochrome f oxidation after a laser flash created oxidised PS I reaction centre (P700) and PC, and decreased somewhat the extent of PC oxidation. The re-reduction of oxidised P700 was more complete than when inhibitor was absent. The data were simulated with reactions which included the putative reduction of cytochrome f by the Rieske centre (FeS) and different rate-coefficients according as to whether inhibitor was bound to the bf complex or not. It was concluded that under the conditions studied the Rieske centre donated electrons to oxidised cytochrome f and plastocyanin with an average rate coefficient of 35 s^–1. This electron transfer was prevented by any of the three inhibitors, which also increased the equilibrium coefficient for the cytochrome f/PC reaction by a maximum factor of two. This increase corresponded to a decrease in the back reaction coefficient and an increase in the forward rate. The equilibrium coefficient for the reduction of oxidised P700 by PC was about 2 in the absence of inhibitor but increased to about 20 in their presence, but only if cytochrome bf complex was additionally present. This was attributed to the transient formation of complexes between P700 with bound plastocyanin, and bf complex. The operative mid-point potential of FeS, if that of cytochrome f is 370 mV, was 390 mV. Deviations in midpoint potentials (P700/plastocyanin) from solution values were attributed to the bound state of the reactants. Estimates were made of the binding coefficient of each of the three inhibitors to p-sites in the cytochrome bf complex in the absence of competing quinol. A stoichiometry of two inhibitors per bf dimer was necessary to cause the above changes in reduction potential of cyt f and PC. A result of one inhibitor per dimer was statistically unlikely, particularly in the case of tridecylstigmatellin.Abbreviations Cyt- cytochrome - DBMIB(H₂)- 2,5-dibromo-3--ethyl-6-isopropyl-p-benzoquinone (reduced) - E _m- midpoint reduction potential of a couple relative to the standard hydrogen electrode - e-t- electron transfer - FeS (or R)- Rieske iron-sulphur centre - HEPES- N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - Mega-9- nonoyl-N-methylglucamide - n-site (Q_r-site)- quinone reduction site in cytochrome bf complex - PC- plastocyanin - p-site (Q_o-site)- quinol oxidation site in cytochrome bf complex - PQ- plastoquinone - PSI- Photosystem I - P700- reaction centre in Photosystem I - TDS- tridecyl stigmatellin     

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.     

The kinetics of reactions around the cytochrome bf complex studied in an isolated system

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10^–7 M^–1 s^–1 applied to 80% of the P700⁺ and c. 0.7×10⁷ M^–1 s^–1 to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10⁷ M^–1 s^–1 (forward), and c. 1.6×10⁷ M^–1 s^–1 (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f ⁺ or plastocyanin⁺ by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f ⁺. Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10⁶ M^–1 s^–1. The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.Abbreviations AQS anthraquinone sulphonate - cyt cytochrome - cyt b-563(H) high-potential cyt b-563 - cyt b-563(L) low potential cyt b-563 - FeS(R) the Rieske protein of the cyt bf complex, containing an Fe₂S₂ centre - PC plastocyanin - PS photosystem - P700 reaction centre in PS I     

Cytochrome bc 1 and b 6 f complexes of photosynthetic membranes

All photosynthetic membranes contain a cytochrome bc ₁ or b ₆ f complex that catalyzes the oxidation of quinols and the reduction of a high-potential electron carrier, such as cytochrome c ₂ or plastocyanin. The cytochrome complex also functions in the translocation of protons across the membrane and as a consequence, establishes the proton motive force that is used for the synthesis of ATP. The structure and function of the cytochrome complexes are first reviewed in this chapter. Amino acid sequence information for almost all of the protein subunits of these complexes is now available, and these allow for a detailed consideration of functional domains in the protein subunits and for a further discussion of the evolution of the cytochrome complex in photosynthetic organisms.     

The pet genes of Rhodospirillum rubrum: cloning and sequencing of the genes for the cytochrome bc1-complex

Summary A cytochrome bc ₁-complex of Rs. rubrum was isolated and the three subunits were purified to homogeneity. The N-terminal amino acid sequence of the purified subunits was determined by automatic Edman degradation. The pet genes of Rhodospirillum rubrum coding for the three subunits of the cytochrome bc ₁-complex were isolated from a genomic library of Rs. rubrum using oligonucleotides specific for conserved regions of the subunits from other organisms and a heterologous probe derived from the genes for the complex of Rb. capsulatus. The complete nucleotide sequence of a 5500 by SalI/SphI fragment is described which includes the pet genes and three additional unidentified open reading frames. The N-terminal amino acid sequence of the isolated subunits was used for the identification of the three genes. The genes encoding the subunits are organized as follows: Rieske protein, cytochrome b, cytochrome c ₁. Comparison of the N-terminal protein sequences with the protein sequences deduced from the nucleotide sequence showed that only cytochrome c ₁ is processed during transport and assembly of the three subunits of the complex. Only the N-terminal methionine of the Rieske protein is cleaved off. The similarity of the deduced amino acid sequence of the three subunits to the corresponding subunits of other organisms is described and implications for structural features of the subunits are discussed.Abbreviations BSA bovine serum albumin - SDS sodium dodecylsulphate - Rs Rhodospirillum - Rb Rhodobacter - Pc Paracoccus - Rps Rhodopseudomonas The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X55387     

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.     

Reduced levels of cytochrome bf complex in transgenic tobacco leads to marked photochemical reduction of the plastoquinone pool,without significant change in acclimation to irradiance

Tobacco transgenics with decreased amounts of the FeS apoprotein were generated using an antisense RNA construct targeted against the nuclear-encoded Rieske FeS protein of cytochrome bf complex [Price et al. (1995) Aust J Plant Physiol 22: 285–297]. FeS phenotypes ranging from intermediate to low were obtained which had 69% and 26% of the Rieske FeS protein of wild type. Similar reductions in the other subunits of cytochrome bf complex, cytochrome f, cytochrome b₅₆₃and the 17 kDa subunit, were demonstrated in the thylakoids of intermediate and low FeS phenotypes. Confirmation that the levels of assembled cytochrome bf in leaves matched the levels of the FeS protein was demonstrated by laser flash-induced redox absorbance changes in leaves, with the extents of cytochrome f oxidation and cytochrome b₅₆₃reduction being equivalent to the decreased amounts of the subunits in isolated thylakoids of the antisense plants. Despite greatly enhanced photochemical reduction of Q_Aand the plastoquinone pool in the antisense plants, light acclimation of the FeS phenotypes to irradiance did not occur. Furthermore, the state 1–state 2 transitions were identical in wild type and antisense plants. Our results suggest that neither Q_Anor the plastoquinone pool acts alone in either the redox control of gene expression or the regulation of light energy distribution between the photosystems. We suggest rather that reduced plastoquinone acting at the inner Q_psite of cytochrome bf complex is involved in molecular redox signalling.     

Assembly of Deletion Mutants of the Rieske Iron–Sulfur Protein into the Cytochrome bc 1 Complex of Yeast Mitochondria

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc ₁ complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc ₁ complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212–16218]. The deletion mutants lacking amino acid residues 55–66 or residues 161–180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161–180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc ₁ complex; however, the protein lacking residues 55–66 was assembled in vitro into the bc ₁ complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55–66. This deletion mutant protein was also assembled into the bc ₁ complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55–66, is not required for assembly of the iron-sulfur protein into the bc ₁ complex; however, this association did not lead to enzymatic activity of the bc ₁ complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.     

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     

Specificity of leaf mitochondrial and chloroplast processing systems for nuclear-encoded precursor proteins

The specificity of the mitochondrial and chloroplast processing enzymes for the nuclear-encoded precursor proteins was investigated. Mitochondrial precursor proteins of the Nicotiana plumbaginifolia and the Neurospora crassa subunits of F₁-ATPase and the Neurospora Rieske FeS precursor protein were processed to the correct mature size by matrix extracts isolated from spinach leaves, yeast, rat liver and beef heart. The mitochondrial extracts failed to process chloroplast precursor proteins of the stromal small subunit of ribulose 1,5-bisphosphate carboxylase and the thylakoid 33 kDa protein of the oxygen-evolving complex. Both mitochondrial F₁ precursors were specifically processed by a soluble stromal extract from chloroplasts. However, no processing of the Rieske FeS precursor protein was observed under the same conditions with the chloroplast extract. The cleavage of the mitochondrial F₁ precursors by the chloroplast extract was shown to be sensitive to the metal chelators EDTA and ortho-phenanthroline. The cleavage site of the mitochondrial F₁ precursor by the chloroplast soluble extract appears to be located at the N-terminus.Abbreviations ATPase adenosine triphosphatase - Rieske FeS non-heme iron sulphur protein of the ubiquinol cytochrome c oxidoreductase complex - Rubisco ribulose 1,5-bisphosphate carboxygenase/oxygenase - RMSF phenylmethylsulphonylfluoride - EDTA ethylenediaminetetraacetic acid     

The cloning and sequencing of Synechococcus sp. PCC 7002 petCA operon: Implications for the cytochrome c-553 binding domain of cytochrome f

The genes encoding the Rieske iron-sulfur protein and cytochrome f from a unicellular, naturally transformable, photoheterotrophic cyanobacterium, Synechococcus sp. PCC 7002, formerly Agmenellum quadruplicatum, have been isolated and sequenced. The two genes were found to be on a single operon, petCA.The Synechococcus sp. PCC 7002 iron-sulfur protein contains 181 amino acids, the conserved putative iron-binding domains CTHLGCV, residues 108–114, and CPCHGS, residues 128–133, no presequence and has a 73% sequence identity to the Nostoc PCC 7906 iron-sulfur protein. The 325 amino acid apocytochrome f sequence contains a 42 amino acid presequence, a CANCH heme binding domain, residues 20–24 from the presumed start of the mature protein, and a predicted hydrophobic membrane-spanning domain, residues 250–269. The mature cytochrome f sequence has a 71.5% sequence identity with Nostoc PCC 7906 cytochrome f and possesses a large (-14) negative charge and low calculated pI of 4.47 compared to higher plant chloroplast sequences. Nine separate domains showing differences in charged residues among cyanobacteria and plants have been identified and the possibility that these domains are involved in the ionic interactions with plastocyanin or cytochrome c-553 is discussed.The sequences reported in this paper have been deposited in the EMBL/Genbank data base (IntelliGenetics, Mountain View, CA, and Eur. Mol. Biol. Lab., Heidelberg) (accession no. M74514).     

Human Cytochrome c Oxidase: Structure, Function, and Deficiency

As the terminal component of the mitochondrial respiratory chain, cytochrome c oxidase plays a vital role in cellular energy transformation. Human cytochrome c oxidase is composed of 13 subunits. The three major subunits form the catalytic core and are encoded by mitochondrial DNA (mtDNA). The remaining subunits are nuclear-encoded. The primary sequence is known for all human subunits and the crystal structure of bovine heart cytochrome c oxidase has recently been reported. However, despite this wealth of structural information, the role of the nuclear-encoded subunits is still poorly understood. Yeast cytochrome c oxidase is a close model of its human counterpart and provides a means of studying the effects of mutations on the assembly, structure, stability and function of the enzyme complex. Defects in cytochrome c oxidase function are found in a clinically heterogeneous group of disorders. The molecular defects that underlie these diseases may arise from mutations of either the mitochondrial or the nuclear genomes or both. A significant number of cytochrome c oxidase deficiencies, often associated with other respiratory chain enzyme defects, are attributed to mutations of mtDNA. Mutations of mtDNA appear, nonetheless, uncommon in early childhood. Pedigree analysis and cell fusion experiments have demonstrated a nuclear involvement in some infantile cases but a specific nuclear genomic lesion has not yet been reported. Detailed analyses of the many steps involved in the biogenesis of cytochrome c oxidase, often pioneered in yeast, offer several starting points for further molecular characterizations of cytochrome c oxidase deficiencies observed in clinical practice.     

Low molecular weight subunits associated with the cytochrome b 6 f complexes from spinach and Chlamydomonas reinhardtii

Cytochrome b ₆ f complexes, prepared from spinach and Chlamydomonas thylakoids, have been examined for their content of low molecular weight subunits. The spinach complex contains two prominent low molecular weight subunits of 3.7 and 4.1 kD while a single prominent component of 4.5 kD was present in the Chlamydomonas complex. An estimation of the relative stoichiometry of these subunits suggests several are present at levels approximating one copy per cytochrome complex. The low molecular weight subunits were purified by reversed phase HPLC and N-terminal sequences obtained. Both the spinach and Chlamydomonas cytochrome complexes contain a subunit that is identified as the previously characterized petG gene product (4.8 kD in spinach and 4.1 kD in Chlamydomonas). A second subunit (3.8 kD in spinach and 3.7 kD in Chlamydomonas) appears to be homologous in the two complexes and is likely to be a nuclear gene product. The possible presence of other low molecular weight subunits in these complexes is also considered.     

Modification of the apparent redox reaction between cytochrome f and the rieske iron-sulfur protein

We have investigated the role of cytochrome f and the Rieske FeS protein in spinach chloroplasts using the quinone analogue 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). UHDBT inhibits electron transport at two different sites in spinach chloroplasts. Fluorescence yield measurements monitoring the redox state of Q, the first stable primary acceptor of Photosystem II, and polarographic measurements of electron transport show that at low concentrations UHDBT inhibits near Q. At higher concentrations UHDBT inhibits at a second site. Electron transfer from durohydroquinone to methyl viologen is inhibited (50% inhibition at 21 μM) but not the reaction dichlorophenolindophenol to methyl viologen. Spectroscopic measurements of the kinetics of cytochrome f show that UHDBT inhibits the dark reduction rate of the cytochrome following a 100 ms flash (50% inhibition at 15 μM). By contrast, the oxidation kinetics of cytochrome f following a single-turnover flash are altered little by UHDBT; the initial rates are indistinguishable, and the half-time increases from 220 μs in the control to 285 μs in the presence of 15 μM UHDBT, largely because the extent of the cytochrome f oxidation is enhanced 1.4-fold in the presence of the inhibitor. In a single-turnover flash in the absence of UHDBT, we observe 38–48% of the total cytochrome f turning over, while in the presence of UHDBT we observe 60–69% of the cytochrome turning over. We interpret these results in terms of a linear rapid donor pool to Photosystem I, FeS → cytochrome f → plastocyanin → P-700, in which UHDBT inhibits by interacting with the Rieske FeS center. We conclude that the enhanced extent of cytochrome f oxidation in the presence of UHDBT is due to the removal of the Rieske FeS center from the rapid donor pool. As a consequence, removal of a single electron from the pool results in a greater cytochrome f oxidation. These results indicate that the Rieske FeS center and cytochrome f equilibrate in a time period comparable to the oxidation time of the cytochrome.     

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.     

The plastid rpoA gene encoding a protein homologous to the bacterial RNA polymerase alpha subunit is expressed in pea chloroplasts

Summary The gene rpoA, encoding a protein homologous to the alpha subunit of RNA polymerase from Escherichia coli has been located in pea chloroplast DNA downstream of the petD gene for subunit IV of the cytochrome b-f complex. Nucleotide sequence analysis has revealed that rpoA encodes a polypeptide of 334 amino acid residues with a molecular weight of 38916. Northern blot analysis has shown that rpoA is co-transcribed with the gene for ribosomal protein S11. A lacZ-rpoA gene-fusion has been constructed and expressed in E. coli. Antibodies raised against the fusion protein have been employed to demonstrate the synthesis of the rpoA gene product in isolated pea chloroplasts. Western blot analysis using these antibodies and antibodies against the RNA polymerase core enzyme from the cyanobacterium, Anabaena 7120, has revealed the presence of the gene product in a crude RNA polymerase preparation from pea chloroplasts.