Günter Hauska

Amino acid identities in the three redox center-carrying polypeptides of cytochromebc1/b6f complexes

The comparison of primary structures is extended to 22 cytochromesb orb6, 12 cytochromesc1 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 reaction center of green sulfur bacteria

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)(3)(PscA)(2)PscB(PscC)(2)PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NAD(+)-reduction is unknown, since a gene equivalent to ferredoxin-NADP(+) reductase is not present in the genome.

Genes and transcripts for the polypeptides of the cytochrome b6/f complex from spinach thylakoid membranes.

Cytochrome b6/f complex was prepared from washed thylakoid membranes by a procedure involving detergent treatment and centrifugation in sucrose gradients. The complex is composed of at least four polypeptide species, cytochrome f which occurs in two variant forms (mol. wt. 34/33 kd), cytochrome b6 (23 kd), the high‐potential Rieske iron‐sulfur protein (19 kd) and a fourth subunit (17 kd) of unknown function. Transcripts for the cytochromes f, b6 and subunit 4 were found in plastid RNA, those for the Rieske iron‐sulfur protein in cytosolic poly(A)+ RNA. Transcripts for cytochrome b6 and subunit 4 are translated in rabbit reticulocyte lysates into products of correct length. The Rieske iron‐sulfur protein and the cytochrome f apoprotein appear to be made as precursors with excess sequences of 7 and 4 kd, respectively. Cytochrome f, cytochrome b6 and subunit 4 are encoded by uninterrupted plastid genes that are located in the large single‐copy region of the circular DNA molecule. Each of these genes is present once per chromosome. Their location and direction of transcription have been determined by hybrid‐selection mapping and by cell‐free transcription/translation of various recombinant DNAs. The genes for cytochrome b6 and for subunit 4 lie near each other, but do not overlap. They are transcribed into a single message. The gene for cytochrome f maps 15 kbp away from this cluster, close to the 3′ end of the gene for the large subunit of ribulosebisphosphate carboxylase/oxygenase, and is transcribed into a separate 4 kb long RNA. All these genes have the same polarities with respect to each other.

Identification of the polypeptides in the cytochromeb6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb6/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, cytochromeb6, 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 cytochromesb6 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 cytochromeb6 was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withEm(1)=−40 mV andEm(2)=−172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.

Purification of membrane‐bound cytochromes and a photoactive P840 protein complex of the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum

A photoactive P840 protein complex and a fraction enriched in cytochrome b from chromatophores of Chlorobium limicola f. thiosulfatophilum are described. The former is identical in pigment composition to the ‘core complex’ of Prostecochloris aestuarii [(1983) Biochim. Biophys. Acta 725, 361–367]. It consists of 3 major polypeptides. The dominating one, of 65 kDa and carrying bacteriochlorophyll, is the P840 reaction center protein. It is probably very similar to the P700 reaction center protein of chloroplasts and cyanobacteria. A 24‐kDa protein could be identified as an ascorbate‐reducible cytochrome c‐550.5. The third polypeptide of 32 kDa might be a Rieske‐type FeS protein. The cytochrome b fraction consists of 2 polypeptides of 42 kDa, representing cytochrome b‐562, and 24 kDa, representing ascorbate‐reducible cytochrome c‐550.5 again. Conditions can be varied to obtain cytochrome b‐562 in pure form. A first characterization of the components is presented.

Vectorial redox reactions of physiological quinones I. Requirement of a minimum length of the isoprenoid side chain

Physiological quinones carrying isoprenoid side chains have been compared with homologues lacking the side chain, for their ability to carry electrons and protons from dithionite to ferricyanide, trapped in liposomes. Six differential observations were made: (1) Plastoquinone and ubiquinones, with a side chain of more than two isoprene units, are by far better mediators than their short-chain homologues. Also other benzoquinones lacking a long side chain are poor catalysts, except dimethyl-methylenedioxy-p-benzoquinone, a highly autooxidizable compound. Tocopherol is a good catalyst. (2) Vitamin K-1 and K-2 are poor mediators compared to vitamin K-3. (3) The reaction catalyzed by quinones carrying long isoprenoid side chains has an about three-fold higher activation energy, irrespective of the catalytic efficiency. (4) The reaction catalyzed by quinones lacking a long side chain follows pseudo first-order kinetics, while the reaction with quinones carrying a long side chain is of apparently higher order. (5) The rate with ubiquinone-1 is increasing pH, while with ubiquinone-9 it is decreasing. (6) The reaction mediated by short-chain quinones seems to be satuarated at lower dithionite concentration. We conclude that isoprenoid quinones are able to translocate electrons and protons in lipid membranes, and that the side chain has a strong impact on the mechanism. This and the relevance of the model reaction for electron and proton transport in photosynthesis and respiration is discussed.

A cytochrome f-b6 complex with plastoquinol-cytochrome c oxidoreductase activity from Anabaena variabilis

A cytochrome f-b6 complex has been isolated from thylakoid membranes of Anabaena variabilis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of four major polypeptides - cytochrome f (molecular mass 31 or 38 kDa, depending on sample preparation for the electrophoresis), cytochrome b6 (molecular mass 22.5 kDa), a band with slightly lower mobility than cytochrome b6 resolved only in gels containing urea, and a 16.5 kDa polypeptide, - plus two small polypeptides below 10 kDa. The complex is devoid of chlorophyll and carotenoids, contains 2 cytochromes b6 per cytochrome f, but no cytochrome b-559. The Rieske Fe-S center is present in substoichiometric amounts of cytochrome f. The complex is active in plastoquinol-cytochrome c oxidoreductase activity, which is specific for electron-accepting proteins with positive net charge at physiological pH. Cytochrome c from horse heart, cytochrome c-553 and plastocyanin, both from A. variabilis, are equally well reduced. The specificity for the electron-donating quinol is less stringent. The sensitivity of the oxidoreductase to various inhibitors is retained during the isolation procedure, 2,5-dibromo-3-methyl-isopropyl-p-benzoquinone being the most efficient. The complex exhibits oxidant-induced reduction of cytochrome b6, and photooxidation of cytochrome f as well as transient photoreduction of cytochrome b6 when illuminated in the presence of plastoquinol, plastocyanin and Photosystem I reaction centers.

Sulfide quinone reductase (SQR) activity in Chlorobium

Membranes of the green sulfur bacterium, Chlorobium limicola f, thiosulfatophilum, catalyze the reduction of externally added isoprenoid quinones by sulfide. This activity is highly sensitive to stigmatellin and aurachins. It is also inhibited by 2‐n‐nonyl‐4‐hydroxyquinoline‐N‐oxide, antimycin, myxothiazol and cyanide. It is concluded that in sulfide oxidizing bacteria like Chlorobium, sulfide oxidation involves a sulfide‐quinone reductase (SQR) similar to the one found in Oscilatoria limnetica [Arieli, B., Padan, E. and Shahak, Y. (1991) J. Biol. Chem. 266. 104–111].

The photosystem I-like P840-reaction center of green S-bacteria is a homodimer.

An operon encoding the P840 reaction center of Chlorobium limicola f.sp.thiosulfatophilum has been cloned and sequenced. It contains two structural genes coding for proteins of 730 and 232 amino acids. The first protein resembles the large subunits of the Photosystem I (PS I) reaction center. Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS I and for the acceptors A(o), A(1) and FeS-center X are conserved. The second protein is related to the PS I subunit carrying the FeS-centers A and B. Since all our efforts to find a gene for a second, large subunit failed, the P840 reaction center probably is homodimeric.

Electrogenic proton translocation by the chloroplast cytochrome b6/f complex reconstituted into phospholipid vesicles

When the cytochrome b 6/f complex from chloroplasts is incorporated into liposomes, reduction of external plastocyanin by plastoquinol is stimulated under uncoupling conditions. An extra H+/e− is ejected from the vesicles during the reaction, in addition to the scalar proton liberated from plastoquinol. This is stimulated by valinomycin/K+ and abolished under uncoupling conditions. Furthermore, the formation of a membrane potential during the reaction, negative inside the vesicles, is observed with the help of carbocyanine dyes. We conclude that the cytochrome b 6/f complex, like the cytochrome bc 1 from mitochondria, functions as an electrogenic proton translocator.