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).     

The gene for the 10 kDa phosphoprotein of photosystem II is located in chloroplast DNA

Nucleotide sequencing of a region of wheat chloroplast DNA between the genes for the 47 kDa chlorophyll a-binding protein of photosystem II (psbB) and cytochrome b-563 (petB) has revealed an open reading frame of 73 codons. This open reading frame has been identified as the gene (psbH) for the 10 kDa phosphoprotein of photosystem II by comparison with the published N-terminal amino acid sequence and amino acid composition of the purified spinach protein. The predicted sequence of the protein shows some homology with the N-terminal region of the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII).     

Deficiency in mRNA splicing in a cytochrome c mutant of neurospora crassa: importance of carboxy terminus for import of apocytochrome c into mitochondria.

Molecular cloning and characterization of cytochrome c cDNA clones of Neurospora crassa wild-type (74A) and a cytochrome c-deficient mutant (cyc1-1) are described. Southern blot analysis of genomic DNA indicates that only one cytochrome c gene exists in the N. crassa genome. The cDNA sequence of the wild-type cytochrome c confirmed the previously determined protein sequence. Sequence analysis of the cyc1-1 cDNA for cytochrome c revealed the presence of a larger open reading frame, owing to the presence of an unspliced intron in the 3' end of the coding region. Splicing of this intron is obviously prevented due to the presence of two base exchanges in the highly conserved intron consensus sequences. Consequently, cyc1-1 synthesizes apocytochrome c with an altered carboxy terminus, 19 amino acids longer than the wild-type cytochrome c, with the final 27 amino acids being of an unrelated sequence. This alteration in the carboxy terminus renders the apocytochrome c incompetent for binding to mitochondria and, consequently, import into mitochondria. Thus, unlike other mitochondrial precursor proteins, where it has been demonstrated that the amino terminus alone is sufficient to target the protein to the mitochondria, an intact carboxy terminus is required for efficient import of apocytochrome c into mitochondria. This is independent confirmation for the view that the import pathway of cytochrome c is unique with respect to all other mitochondrial proteins studied to date.     

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants

Cytochrome b559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of cytochrome b559 mutations on its midpoint redox potential and on the reduction of the cytochrome b559 by the plastoquinone pool (or Q_B). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of cytochrome b559 have been investigated. All the mutations led to a destabilization of the high potential form of the cytochrome b559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the cytochrome b559 showing that under conditions of a non-functional water oxidation system, once the cytochrome is oxidized by P680⁺, the yield of its reduction by Q_B or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of cytochrome b559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.     

Concentration and function of membrane-bound cytochromes in cyanobacterial heterocysts

Membranes isolated from heterocysts and vegetative cells of Anabaena 7120 were assayed for content of cytochrome f, cytochrome b-563, cytochrome b-559_HP, cytochrome b-559_LP, and cytochrome aa₃ by use of reduced-minus-oxidized difference spectra. The level of cytochrome aa₃ in heterocyst membranes was 4 to 100 times higher than that in vegetative cells of Anabaena 7120 or other species of cyanobacteria. Heterocyst membranes lack cytochrome b-559_HP but contain cytochrome b-559_LP (E_m7.5 = +77 millivolts, n = 1) at approximately the same concentration as cytochrome f. The role of cytochrome b-559_LP in the hydrogenase-dependent electron transfer pathway was investigated with the inhibitor 2-(n-heptyl)-4-hydroxyquinoline N-oxide which blocks electron flow from hydrogenase to acceptors reacting with the plastoquinone pool. Addition of inhibitor elicited no change in the reduction level of cytochrome b-559_LP indicating that this cytochrome is not directly involved in this pathway.     

The complete amino-acid sequence of the rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis

Summary SummarySeveral cDNA clones encoding the entire Rieske FeS-precursor protein of the chloroplast cytochrome b ₆ f-complex have been isolated by high density plaque immunoscreening of a phage lambda gt11 cDNA expression library, made from poly A⁺-RNA of spinach seedlings. The identity of the cDNAs has been confirmed by N-terminal amino acid sequencing of the purified protein. The nucleotide sequence indicates a protein of 247 amino acid residues including a putative transit sequence of 68 amino acids corresponding to molecular masses of 26.3 kDa (precursor) and 18.8 kDa (mature protein; 179 amino acid residues). Alignteins of the sequence with sequences from Rieske FeS-proteins of respiratory electron transport chains, two of bacterial and three of mitochondrial origin, shows little sequence homology, but remarkable similarity in secondary structure including a putative N-terminal transmembrane segment of about 25 residues and the peptides CTHLGCV and CPCHGS in the C-terminal region of the protein that are involved in the binding of the Fe₂S₂-cluster.     

CCS5, a Thioredoxin-like Protein Involved in the Assembly of Plastid c-Type Cytochromes

The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c₆ in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b₆f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c₆ in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.     

Processing of the 5′-UTR and existence of protein factors that regulate translation of tobacco chloroplast psbN mRNA

The chloroplast psbB operon includes five genes encoding photosystem II and cytochrome b ₆ /f complex components. The psbN gene is located on the opposite strand. PsbN is localized in the thylakoid and is present even in the dark, although its level increases upon illumination and then decreases. However, the translation mechanism of the psbN mRNA remains unclear. Using an in vitro translation system from tobacco chloroplasts and a green fluorescent protein as a reporter protein, we show that translation occurs from a tobacco primary psbN 5′-UTR of 47 nucleotides (nt). Unlike many other chloroplast 5′-UTRs, the psbN 5′-UTR has two processing sites, at ?39 and ?24 upstream from the initiation site. Processing at ?39 enhanced the translation rate fivefold. In contrast, processing at ?24 did not affect the translation rate. These observations suggest that the two distinct processing events regulate, at least in part, the level of PsbN during development. The psbN 5′-UTR has no Shine–Dalgarno (SD)-like sequence. In vitro translation assays with excess amounts of the psbN 5′-UTR or with deleted psbN 5′-UTR sequences demonstrated that protein factors are required for translation and that their binding site is an 18 nt sequence in the 5′-UTR. Mobility shift assays using 10 other chloroplast 5′-UTRs suggested that common or similar proteins are involved in translation of a set of mRNAs lacking SD-like sequences.     

A pathway for the reduction of cytochrome b-559 by Photosystem II in chloroplasts

Cytochrome b-559, which is normally reduced in the dark, was oxidized by preillumination in the presence of N-methyl-phenazonium methosulfate with low intensity far-red light. The average half-time for the photoreduction of oxidized cytochrome b-559 by a long actinic flash ranged from 90 to 110 ms. In the presence of 0.25 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea the half-time for the photoreduction increased to 230 ms although the extent of the absorbance increase was unchanged. Under similar conditions inhibition of electron transport by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and the increase in the chlorophyll fluorescence show that a large fraction of the Photosystem II reaction centers are blocked. These results are consistent with the concept that electrons are shared between different photosynthetic units by a common pool of plastoquinone and imply that the principle pathway for the reduction of cytochrome b-559 by Photosystem II occurs through plastoquinone. In the presence of the uncoupler gramicidin which stimulates non-cyclic electron transport, the rate of photoreduction of cytochrome b-559 is slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 180}\text{ms}$), from which it is inferred that cytochrome b-559 competes with cytochrome f for electrons out of this pool. Comparison of cytochrome b-559 photoreduction and electron transport rates using untreated and KCN-treated chloroplasts indicate that, under conditions of basal electron transport from water to ferricyanide, approximately one-fifth of the electrons from Photosystem II go through cytochrome b-559 to ferricyanide. Further support for this pathway is provided by a comparison of the effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (dibromothymoquinone) on the rates of reduction of cytochrome b-559 and ferricyanide.     

Arabidopsis thaliana sequence analysis confirms the presenceof cyt b-561 in plants: Evidence for a novel protein family

Recent advances in the Arabidopsis sequencing project has elucidated the presence of two genes Atb561-A and Atb561-B that show limited homology to the DNA sequence encoding for the mammalian chromaffin granule cytochrome b-561 (cyt b-561). Detailed analysis of the structural features and conserved residues reveals, however, that the structural homology between the presumptive Arabidopsis proteins and the animal proteins is very high. All proteins are hydrophobic and show highly conserved transmembrane helices. The presumably heme-binding histidine residues in the plant and animal sequences as well as the suggested binding site for the electron acceptor, monodehydroascorbate, are strictly conserved. In contrast, the suggested electron donor (ascorbate) binding site is not very well conserved between the plant and animal sequences questioning the function of this motif. Sequence analysis of the Atb561-B gene demonstrates a different splicing than that initially predicted in silico resulting in a protein with nine extra amino acids and a significantly higher homology to the other cyt b-561 sequences. The homology between the plant and animal sequences is further supported by the strong similarity between a number of biochemical properties of the chromaffin cyt b-561 and the cyt b-561 isolated from bean hook plasma membranes. Since the mammalian cyt b-561 is considered specific to neuroadrenergic tissues, the identification of a closely related homologue in an aneural organism demonstrates that these proteins constitute a new class of widely occurring membrane proteins. Both the plant and animal cyt b-561 are involved in transmembrane electron transport using ascorbate as an electron donor. The similarity between these proteins therefore suggests, for the first time, that this transport supports a number of different cell physiological processes. An evolutionary relationship between the plant and animal proteins is presented.     

Amino acid composition of the 9 kDa phosphoprotein of pea thylakoids

The 9 kDa phosphoprotein of pea thylakoids was isolated by electroelution from SDS-polyacrylamide gels and its amino acid composition determined. The result is at variance with the amino acid compositions predicted from published nucleotide sequences of the genes for apocytochrome b-559 and for CFo subunit III. The amino acid composition of the 9 kDa phosphoprotein resembles that of the 25 kDa light-harvesting chlorophyll a/b protein (LHC-II). We propose that the 9 kDa polypeptide is a chlorophyll-binding protein of photosystem II, that it functions as a link in excitation energy transfer between LHC-II and the reaction centre, and that its phosphorylation regulates excitation energy distribution by means of mutual electrostatic repulsion between itself and phosphorylated LHC-II.     

Assembly of a transmembrane b-Type cytochrome is mainly driven by transmembrane helix interactions

Folding, assembly and stability of α-helical membrane proteins is still not very well understood. Several of these membrane proteins contain cofactors, which are essential for their function and which can be involved in protein assembly and/or stabilization. The effect of heme binding on the assembly and stability of the transmembrane b-type cytochrome b₅₅₉^′ was studied by fluorescence resonance energy transfer. Cytochrome b₅₅₉^′ consists of two monomers of a 44 amino acid long polypeptide, which contains one transmembrane domain. The synthesis of two variants of the b₅₅₉^′ monomer, each carrying a specific fluorescent dye, allowed monitoring helix-helix interactions in micelles by resonance energy transfer. The measurements demonstrate that the transmembrane peptides dimerize in detergent in the absence and presence of the heme cofactor. Cofactor binding only marginally enhances dimerization and, apparently, the redox state of the heme group has no effect on dimerization.     

Stoichiometry of components in the photosynthetic oxygen evolution system of Photosystem II particles prepared with Triton X-100 from spinach chloroplasts

The stoichiometry of the proteins of the photosynthetic oxygen evolution system and of the electron transport components in Photosystem II particles prepared with Triton X-100 from spinach chloroplasts were determined. Per about 220 chlorophyll molecules, there were one reaction center II, one molecule each of the 33, 24 and 18 kDa proteins, four Mn atoms, two cytochromes b-559 (one high-potential, the other low-potential), and 3.5 plastoquinone-9 molecules, but practically no cytochrome b-563, cytochrome f, phylloquinone, α-tocopherol or α-tocopherylquinone.     

Evolutionary Persistence of the Molybdopyranopterin-Containing Sulfite Oxidase Protein Fold

Summary: The importance of molybdoenzymes is exemplified both by the debilitating and fatal human diseases caused by their deficiency and by their persistence throughout evolution. Here, we show that the protein fold of the molybdopyranopterin-containing domain of sulfite oxidase (the SUOX fold) can be found in all three domains of life. Analyses of sequence data and protein structure comparisons (secondary structure matching) show that the SUOX fold is found in enzymes that have quite distinct macromolecular architectures comprising one or more domains and sometimes subsidiary subunits. These are summarized as follows: (i) animal SUOXs that contain an N-terminal cytochrome b₅ domain and an SUOX fold fused to a C-terminal dimerization domain; (ii) plant SUOX that contains an SUOX fold fused to a C-terminal dimerization domain; (iii) the YedY protein from Escherichia coli, which comprises only the SUOX fold; (iv) the sulfite dehydrogenase from Starkeya novella that contains the SUOX fold, a dimerization domain, and an additional c-type cytochrome subunit; and (v) the plant-type nitrate reductases, exemplified by that of Pichia angusta, that contain an N-terminal SUOX fold, a dimerization domain, a cytochrome b₅ domain, and a C-terminal NADH binding flavin adenine dinucleotide-containing domain. We used the primary sequences of the proteins containing an SUOX fold to mine 559 sequences of related proteins. A phylogeny of a nonredundant subset of these sequences was generated, and the resultant clades were categorized by sequence motif analyses in the context of the available protein structures. Based on the motif analyses, cladistics, and domain conservations, we are able to postulate a plausible pathway of SUOX fold enzyme evolution.     

Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b 6 f 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.     

Electron Transport Reactions in Grana Preparations from Spinach Chloroplasts

Fraction 2 (grana-stack) particles prepared with the French press showed absorbance changes, at room temperature and with sodium ascorbate and methyl-viologen, that were produced by the oxidation of cytochrome b-559. This oxidation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and sensitized by system II of photosynthesis. The oxidation is too slow to account for the rates of the Hill reaction that have been observed with nicotinamide-adenine dinucleotide phosphate (NADP⁺). It appears that this cytochrome is not functioning in the main pathway of electron transport. In the presence of 2,3,5,6-tetramethyl-p-phenylene-diamine (DAD) and ascorbate, light-induced oxidation of cytochrome f took place within 3 msec (or faster) in the grana-stack particles. Treatment with the detergent Triton X-100 disrupted this rapid cytochrome f oxidation as well as the oxidation of cytochrome b-559. Subsequent plastocyanin addition did not restore the rapid oxidation of cytochrome f (nor of cytochrome b-559) but only slow changes of cytochrome f. In view of the fact that these particles contain almost no plastocyanin, it is unlikely that plastocyanin functions in electron transport between cytochrome f and P-700 in the particles derived from the grana-stack regions of the chloroplast.