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• W1986201304 abstract "The lumen segment of cytochrome fconsists of a small and a large domain. The role of the small domain in the biogenesis and stability of the cytochromeb6 f complex and electron transfer through the cytochrome b6 f complex was studied with a small domain deletion mutant in Chlamydomonas reinhardtii. The mutant is able to grow photoautotrophically but with a slower rate than the wild type strain. The heme group is covalently attached to the polypeptide, and the visible absorption spectrum of the mutant protein is identical to that of the native protein. The kinetics of electron transfer in the mutant were measured by flash kinetic spectroscopy. Our results show that the rate for the oxidation of cytochrome f was unchanged (t = 12 = ∼100 μs), but the half-time for the reduction of cytochrome f is increased (t 12 = 32 ms; for wild type,t 12 = 2.1 ms). Cytochromeb6 reduction was slower than that of the wild type by a factor of approximately 2 (t 12 = 8.6 ms; for wild type, t 12 = 4.7 ms); the slow phase of the electrochromic band shift also displayed a slower kinetics (t 12 = 5.5 ms; for wild type,t 12= 2.7 ms). The stability of the cytochromeb6 f complex in the mutant was examined by following the kinetics of the degradation of the individual subunits after inhibiting protein synthesis in the chloroplast. The results indicate that the cytochromeb6 f complex in the small domain deletion mutant is less stable than in the wild type. We conclude that the small domain is not essential for the biogenesis of cytochromef and the cytochromeb6 f complex. However, it does have a role in electron transfer through the cytochromeb6 f complex and contributes to the stability of the complex. The lumen segment of cytochrome fconsists of a small and a large domain. The role of the small domain in the biogenesis and stability of the cytochromeb6 f complex and electron transfer through the cytochrome b6 f complex was studied with a small domain deletion mutant in Chlamydomonas reinhardtii. The mutant is able to grow photoautotrophically but with a slower rate than the wild type strain. The heme group is covalently attached to the polypeptide, and the visible absorption spectrum of the mutant protein is identical to that of the native protein. The kinetics of electron transfer in the mutant were measured by flash kinetic spectroscopy. Our results show that the rate for the oxidation of cytochrome f was unchanged (t = 12 = ∼100 μs), but the half-time for the reduction of cytochrome f is increased (t 12 = 32 ms; for wild type,t 12 = 2.1 ms). Cytochromeb6 reduction was slower than that of the wild type by a factor of approximately 2 (t 12 = 8.6 ms; for wild type, t 12 = 4.7 ms); the slow phase of the electrochromic band shift also displayed a slower kinetics (t 12 = 5.5 ms; for wild type,t 12= 2.7 ms). The stability of the cytochromeb6 f complex in the mutant was examined by following the kinetics of the degradation of the individual subunits after inhibiting protein synthesis in the chloroplast. The results indicate that the cytochromeb6 f complex in the small domain deletion mutant is less stable than in the wild type. We conclude that the small domain is not essential for the biogenesis of cytochromef and the cytochromeb6 f complex. However, it does have a role in electron transfer through the cytochromeb6 f complex and contributes to the stability of the complex. iron-sulfur redox center carbonylcyanidep-trifluoromethoxyphenylhydrazone 2-n-heptyl-4-hydroxyquinoline-N-oxide polymerase chain reaction base pair(s) Functioning as a plastoquinone-plastocyanin oxidoreductase, the cytochrome b6 f complex transfers electrons from Photosystem II to Photosystem I in the photosynthetic electron transfer chain of all oxygen-evolving organisms. Cytochrome f is one of the four redox centers and the largest subunit (31 kDa) in the cytochromeb6 f complex. It transfers electrons from the Rieske FeS1 protein to plastocyanin. The biogenesis of cytochrome f and the assembly of the cytochrome b6 fcomplex involve a complicated process (1Wollman F.-A. Minai L. Nechushtai R. Biochim. Biophys. Acta. 1999; 1411: 21-85Crossref PubMed Scopus (217) Google Scholar, 2Hippler M. Redding K. Rochaix J.-D. Biochim. Biophys. Acta. 1998; 1367: 1-62Crossref PubMed Scopus (58) Google Scholar): starting from the synthesis of the cytochrome f precursor to the translocation of this precursor through the thylakoid membrane, the processing of the precursor to give a mature protein, the covalent attachment of heme to the apoprotein, and the assembly of cytochrome f with the other subunits to form a functional cytochromeb6 f complex. Cytochrome fis required for the assembly of the cytochromeb6 f complex because when thepetA gene encoding cytochrome f in the chloroplast genome was disrupted, there was no assembled cytochromeb6 f complex, and the resulting mutant could not grow photoautotrophically (3Kuras R. Wollman F.-A. EMBO J. 1994; 13: 1019-1027Crossref PubMed Scopus (155) Google Scholar, 14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Biochemical evidence (4Gray J. Photosynth. Res. 1992; 34: 359-374Crossref PubMed Scopus (75) Google Scholar) and the three-dimensional structure (5Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 6Martinez S.E. Huang D. Ponamarev M. Cramer W.A. Smith J.L. Protein Sci. 1996; 5: 1081-1092Crossref PubMed Scopus (132) Google Scholar, 7Chi Y.-I. Huang L.-S. Zhang Z. Fernández-Velasco J.G. Berry E.A. Biochemistry. 2000; 39: 7689-7701Crossref PubMed Scopus (45) Google Scholar, 8Carrell C.J. Schlarb B.G. Bendall D.S. Howe C.J. Cramer W.A. Smith J.L. Biochemistry. 1999; 38: 9590-9599Crossref PubMed Scopus (72) Google Scholar) revealed that there are three domains in cytochrome f: a C-terminal transmembrane span and a small and a large domain. Serving as a transmembrane span, the C-terminal hydrophobic region of cytochrome f (from Gln253 to Phe286 in Chlamydomonas reinhardtii) (9Büschlen S. Choquet R. Kuras R. Wollman F.-A. FEBS Lett. 1991; 284: 257-262Crossref PubMed Scopus (47) Google Scholar, 10Matsumoto T. Matsuo M. Matsuda Y. Plant Cell Physiol. 1991; 32: 863-872Google Scholar, 11Bertsch J. Malkin R. Plant Mol. Biol. 1991; 17: 131-133Crossref PubMed Scopus (8) Google Scholar) anchors the protein to the thylakoid membrane. Deletion of this region gave rise to a soluble form of cytochrome f (12Kuras R. Wollman F.-A. Joliot P. Biochemistry. 1995; 34: 7468-7475Crossref PubMed Scopus (40) Google Scholar) that was transported to the lumen and was capable of transferring electrons to plastocyanin, but again there was no assembled cytochrome b6 f complex (12Kuras R. Wollman F.-A. Joliot P. Biochemistry. 1995; 34: 7468-7475Crossref PubMed Scopus (40) Google Scholar). Localized in the lumen, the soluble segment has an elongated structure, with a small and a large domain (Refs. 5Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 6Martinez S.E. Huang D. Ponamarev M. Cramer W.A. Smith J.L. Protein Sci. 1996; 5: 1081-1092Crossref PubMed Scopus (132) Google Scholar, 7Chi Y.-I. Huang L.-S. Zhang Z. Fernández-Velasco J.G. Berry E.A. Biochemistry. 2000; 39: 7689-7701Crossref PubMed Scopus (45) Google Scholar, 8Carrell C.J. Schlarb B.G. Bendall D.S. Howe C.J. Cramer W.A. Smith J.L. Biochemistry. 1999; 38: 9590-9599Crossref PubMed Scopus (72) Google Scholar; see Fig. 1). A lysine patch was found in cytochrome f (5Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 6Martinez S.E. Huang D. Ponamarev M. Cramer W.A. Smith J.L. Protein Sci. 1996; 5: 1081-1092Crossref PubMed Scopus (132) Google Scholar, 7Chi Y.-I. Huang L.-S. Zhang Z. Fernández-Velasco J.G. Berry E.A. Biochemistry. 2000; 39: 7689-7701Crossref PubMed Scopus (45) Google Scholar) from higher plants and C. reinhardtii and was postulated to be involved in the interaction with plastocyanin (5Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). Mutations within the large domain generated interference with the electron transfer (13Kuras R. Büschlen S. Wollman F.-A. J. Biol. Chem. 1995; 270: 27797-27803Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 15Soriano G.M. Ponamarev M.V. Tae G.-S. Cramer W.A. Biochemistry. 1996; 35: 14590-14598Crossref PubMed Scopus (72) Google Scholar, 16Comolli L.R. Zhou J Linden T. Breitling R. Flores J. Hung T. Jamshidi A. Huang L.-S. Fernández-Velasco J.G. Garab G. Photosynthesis: Mechanisms and Effects. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 1589-1592Google Scholar, 17Soriano G.M. Ponamarev M.V. Piskorowski R.A. Cramer W.A. Biochemistry. 1998; 37: 15120-15128Crossref PubMed Scopus (56) Google Scholar, 18Ponamarev M.V. Cramer W.A. Biochemistry. 1998; 37: 17199-17208Crossref PubMed Scopus (61) Google Scholar, 19Baymann F. Zito F. Kuras R. Minai L. Nitschke W. Wollman F.-A. J. Biol. Chem. 1999; 274: 22957-22967Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 20Gong X.-S. Wen J.Q. Gray J. Eur. J. Biochem. 2000; 267: 1732-1742Crossref PubMed Scopus (20) Google Scholar, 21Gong X.-S. Wen J.Q. Fisher N.E. Young S. Howe C.J. Bendall D.S. Gray J. Eur. J. Biochem. 2000; 267: 3461-3468Crossref PubMed Scopus (39) Google Scholar), the proton translocation (18Ponamarev M.V. Cramer W.A. Biochemistry. 1998; 37: 17199-17208Crossref PubMed Scopus (61) Google Scholar), and the maturation of cytochromef (13Kuras R. Büschlen S. Wollman F.-A. J. Biol. Chem. 1995; 270: 27797-27803Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 19Baymann F. Zito F. Kuras R. Minai L. Nitschke W. Wollman F.-A. J. Biol. Chem. 1999; 274: 22957-22967Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). When mutants were isolated in which heme attachment was altered in the large domain of cytochrome f, the assembly of the cytochrome b6 fcomplex and the insertion of the heme group were disrupted (13Kuras R. Büschlen S. Wollman F.-A. J. Biol. Chem. 1995; 270: 27797-27803Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Although these results shed light on the functioning of C-terminal transmembrane span and the large domain of cytochrome f, the role of the small domain remained ambiguous. The small domain of cytochrome f consists of amino acid residues from Asn172 to Leu228 (9Büschlen S. Choquet R. Kuras R. Wollman F.-A. FEBS Lett. 1991; 284: 257-262Crossref PubMed Scopus (47) Google Scholar, 10Matsumoto T. Matsuo M. Matsuda Y. Plant Cell Physiol. 1991; 32: 863-872Google Scholar, 11Bertsch J. Malkin R. Plant Mol. Biol. 1991; 17: 131-133Crossref PubMed Scopus (8) Google Scholar), approximately the difference in size between the lumen-soluble segment of cytochrome f and the extramembrane domain of mitochondrial cytochrome c1 (5Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). In comparing the primary sequences of cytochrome f from other organisms, the small domain of cytochrome f is less conserved than the large domain (4Gray J. Photosynth. Res. 1992; 34: 359-374Crossref PubMed Scopus (75) Google Scholar). Only a few amino acid residues within its sequence are identical among all of the sequences found. From the crystal structure of cytochrome f (5Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 6Martinez S.E. Huang D. Ponamarev M. Cramer W.A. Smith J.L. Protein Sci. 1996; 5: 1081-1092Crossref PubMed Scopus (132) Google Scholar, 7Chi Y.-I. Huang L.-S. Zhang Z. Fernández-Velasco J.G. Berry E.A. Biochemistry. 2000; 39: 7689-7701Crossref PubMed Scopus (45) Google Scholar, 8Carrell C.J. Schlarb B.G. Bendall D.S. Howe C.J. Cramer W.A. Smith J.L. Biochemistry. 1999; 38: 9590-9599Crossref PubMed Scopus (72) Google Scholar), we can see that the backbone of the small domain is distinguishable from the large domain (Fig. 1). Mutations of amino acid residues within the small domain did not reveal any significant difference from the wild type protein in vivo (15Soriano G.M. Ponamarev M.V. Tae G.-S. Cramer W.A. Biochemistry. 1996; 35: 14590-14598Crossref PubMed Scopus (72) Google Scholar, 16Comolli L.R. Zhou J Linden T. Breitling R. Flores J. Hung T. Jamshidi A. Huang L.-S. Fernández-Velasco J.G. Garab G. Photosynthesis: Mechanisms and Effects. Kluwer Academic Publishers, Dordrecht, The Netherlands1998: 1589-1592Google Scholar, 17Soriano G.M. Ponamarev M.V. Piskorowski R.A. Cramer W.A. Biochemistry. 1998; 37: 15120-15128Crossref PubMed Scopus (56) Google Scholar). These analyses raised the question about the role of the small domain of cytochrome f in the biogenesis and functioning of the cytochrome b6 f complex. In a comparison of the structure of the cytochromeb6 f complex (5Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 6Martinez S.E. Huang D. Ponamarev M. Cramer W.A. Smith J.L. Protein Sci. 1996; 5: 1081-1092Crossref PubMed Scopus (132) Google Scholar, 7Chi Y.-I. Huang L.-S. Zhang Z. Fernández-Velasco J.G. Berry E.A. Biochemistry. 2000; 39: 7689-7701Crossref PubMed Scopus (45) Google Scholar, 8Carrell C.J. Schlarb B.G. Bendall D.S. Howe C.J. Cramer W.A. Smith J.L. Biochemistry. 1999; 38: 9590-9599Crossref PubMed Scopus (72) Google Scholar, 22Mosser G. Breyton C. Olofsson A. Popot J.-L. Right J.-L. J. Biol. Chem. 1997; 272: 20263-20268Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 23Bron P. Lacapère J.-J. Breyton C. Mosser G. J. Mol. Biol. 1999; 287: 117-126Crossref PubMed Scopus (21) Google Scholar) with the analogous cytochrome bc1 complex from mitochondria (24Xia D., Yu, C.-A. Kim H. Xia J.-Z. Kachurin A.M. Zhang L., Yu, L. Deisenhofer J. Science. 1997; 277: 60-66Crossref PubMed Scopus (873) Google Scholar, 25Zhang Z. Huang L. Shulmeister V.M. Chi Y.-I. Kim K.K. Hung L.-W. Crofts A.R. Berry E.A. Kim S.-H. Nature. 1998; 392: 677-684Crossref PubMed Scopus (939) Google Scholar, 26Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1069) Google Scholar), Soriano et al. (27Soriano G.M. Ponamarev M.V. Carrell C.J. Xia D. Smith J.L. Cramer W.A. J. Bioenerg. Biomembr. 1999; 31: 201-213Crossref PubMed Scopus (43) Google Scholar) speculated that the small domain of cytochrome f might function like the subunit VIII in the cytochrome bc1 complex to provide structural stability to the complex. Recently, the crystal structure of cytochrome f from C. reinhardtiirevealed that cytochrome f could exist in dimeric form (7Chi Y.-I. Huang L.-S. Zhang Z. Fernández-Velasco J.G. Berry E.A. Biochemistry. 2000; 39: 7689-7701Crossref PubMed Scopus (45) Google Scholar), and amino acid residues from the small domain are found to be involved in the hydrogen bond formation in the interface of this dimer. However, the relationship of this dimer to the structure of cytochromef in vivo is not clear. We were interested in defining the role of the small domain of cytochrome f in the biogenesis and the functioning of cytochrome f and the cytochromeb6 f complex. To address this question, we have made a small domain deletion mutant of cytochromef in C. reinhardtii. The mutant is able to grow photoautotrophically and has an assembled cytochromeb6 f complex. Our studies showed that the electron transfer through the cytochromeb6 f complex can occur in the mutant but at a slower rate. In addition, the mutant cytochromeb6 f complex was not as stable as that in the wild type strain. C. reinhardtiiwild type (cc-125), cytochrome f small domain deletion mutant (pFSSD), and ΔpetA mutant (14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) strains were maintained on TAP plates (28Harris, E. (1989) The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use, pp. 25–63 and 593–594, Academic Press Inc., San Diego, CA.Google Scholar) under dim light (2 μmol of photons m−2 s−1). For the photoautotrophic growth of strains, the wild type and the pFSSD mutant were grown on HS plates (28Harris, E. (1989) The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use, pp. 25–63 and 593–594, Academic Press Inc., San Diego, CA.Google Scholar) under medium light (35 μmol of photons m−2 s−1). Liquid cultures of the wild type strain and the pFSSD mutant were carried out in HS medium bubbled with 3% CO2 under 35 μmol of photons m−2s−1 light intensity. Bacteria Escherichia coliDH5α strain was used for DNA manipulations. PCR was performed as described previously (20Gong X.-S. Wen J.Q. Gray J. Eur. J. Biochem. 2000; 267: 1732-1742Crossref PubMed Scopus (20) Google Scholar) to make the construct (Fig. 2). The following are the sequences of the following primers: primer I, 5′-CAACTGGAATCCCCTTATAG-3′ (located 50 bp upstream of petAgene); primer II, 5′-CGCTACGTAAATAGTGTTGTTTGA-3′ (the bold codon encodes Tyr171); primer III, 5′-GCCTACGTA ACAAACAACAACCCTAACGTTGG-3′ (the bold codon encodes Thr229); and primer IV, 5′-GTAGGAGCTGCACAGCAGCC-3′ (located 13 bp downstream from thepetA gene). The underlined sequence is the SnaBI restriction site. The PCR template was the plasmid pJB101ΔH containing the petA gene (14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). A 700-bp fragment I was obtained by PCR using primers I and II. A new SnaBI site was introduced at the 3′-end of the fragment. Fragment II with a length of 220 bp was obtained by PCR using primers III and IV. A newSnaBI site was also added to the 5′-end of fragment II. Fragment I was cut with HindIII and cloned into pUC19 plasmid cut with HindIII and HincII to give pUCFI. Fragment II was cloned into pUC19 cut with HincII to give pUCFII. For the pUCFII, we selected the construct with an orientation where the SnaBI site is adjacent to theHindIII site from the pUC19 polylinker region. The correct sequences of the inserts were confirmed by sequencing in an Applied Biosystems DNA sequencer 377. pUCFII was then digested withSnaBI and EcoRI to give a 220-bp fragment, which was ligated with pUCFI cut with the same enzyme to give pUCFSD. pUCFSD was digested with HindIII and AflII. The insertion was cloned into pJB101ΔH plasmid digested with the same restriction enzyme to give pJBFSD. To make the chloroplast transformation construct pFSSD, the petA gene sequence from pADFI283ST (3Kuras R. Wollman F.-A. EMBO J. 1994; 13: 1019-1027Crossref PubMed Scopus (155) Google Scholar) was replaced with small domain deletion petAmutant sequence from pJBFSD by digesting both plasmids withBglII and EcoRV. In the pFSSD construct the sequence of petA gene, which encodes a small domain of cytochrome f from Gln172 to Leu228, was deleted (57-amino acid sequence). An extra codon for valine was added in the petA gene sequence of the small domain deletion mutant between the codon for Tyr171 and the codon for Thr229 that resulted from the addition of a newSnaBI site. The ΔpetA strain was transformed as described previously (14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) using the biolistic particle delivery system (PDS-1000/He; Bio-Rad). 250 ml of TAP medium was inoculated with the ΔpetA strain and grown for 3 days under dim light. 600 ml of fresh TAP medium was inoculated to ∼1 × 105cells/ml. Cells grown to ∼2 × 106 cells/ml in the presence of 0.5 mm fluorodeoxyuridine were harvested by centrifugation and resuspended to a concentration of ∼1 × 108 cells/ml. 1 ml of the cell suspension was mixed with an equal volume of (premelted and incubated at 42 °C) 0.2% agar in TAP, and 0.7 of the mixture was spread onto two preprepared TAP plates. 4 μg of pFSSD DNA was precipitated onto 0.3 mg of gold particles, which were used to bombard the cells on the plates. The bombarded cells were transferred to TAP plates containing 150 μg/ml spectinomycin and incubated under dim light at 25 °C. PCR was performed to select the transformants as described previously by Berthold et al.(29Berthold D.A. Best B.A. Malkin R. Plant Mol. Biol. Rep. 1993; 11: 338-344Crossref Scopus (19) Google Scholar). C. reinhardtii DNA was prepared by centrifuging cells from 50 ml of culture. The cells were resuspended in 500 μl of 2% hexadecyltrimethylammonium bromide, 100 mmTris-Cl, pH 8, 1.4 m NaCl, 20 mm EDTA, 2% β-mercaptoethanol and incubated at 65 °C for 60 min. The solution was extracted three times with phenol/chloroform/isoamyl alcohol (24:24:1 v/v/v). The DNA was precipitated with 0.7 volumes of isopropyl alcohol. After digesting the DNA with EcoRV andHindIII, Southern blot hybridization was carried out as described previously (14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Prior to electrophoresis for Western blotting, protein samples were prepared according to Berthold et al. (30Berthold D.A. Schmidt C.L. Malkin R. J. Biol. Chem. 1995; 270: 29293-29298Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The protein samples were separated on SDS-polyacrylamide gel with a 15% resolving/5% stacking gel and transferred to nylon membranes. Western blotting was carried out following manufacturer's protocol (ECL Western blotting; Amersham Pharmacia Biotech). Rabbit sera containing polyclonal antibodies generated against spinach cytochromef, spinach subunit IV, C. reinhardtii Rieske FeS protein (generously provided by Dr. C. de Vitry, Institut de Biologie Physico-Chimique, France), and a synthetic peptide conjugate (corresponding to C. reinhardtii cytochromeb6, generously provided by Dr. W. Cramer, Purdue University) were used at a 1:10,000 dilution. Heme peroxidase activity was detected withN,N,N′,N′-tetramethylbenzidine and H2O2 (31Goodhew C.F. Brown K.R. Pettigrew G.W. Biochim. Biophys. Acta. 1986; 852: 288-294Crossref Scopus (155) Google Scholar) The rate of oxygen evolution was measured at 25 °C in an Oxygraph System (Hansatech) according to the manufacturer's instructions. C. reinhardtii cells were resuspended at a chlorophyll concentration of 10 μg/ml in HS medium supplemented with 10 mm sodium bicarbonate (14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Kinetic measurements were performed on autolysin-treated cells to form a homogenous single-cell suspension, which also allows easy access of inhibitors and uncouplers. Autolysin was prepared as described (28Harris, E. (1989) The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use, pp. 25–63 and 593–594, Academic Press Inc., San Diego, CA.Google Scholar). The pellet of C. reinhardtii cells was resuspended with autolysin solution and incubated at room temperature for 15 min. Cells were then washed twice with HS liquid medium. A home-built single beam kinetic spectrophotometer with microsecond time resolution was used as described previously (14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Flash-induced spectroscopy was done at 25 °C under anaerobic conditions maintained by argon flux. Cells were suspended in HS medium, pH 6.8 (28Harris, E. (1989) The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use, pp. 25–63 and 593–594, Academic Press Inc., San Diego, CA.Google Scholar), at a chlorophyll concentration of 30 μg/ml. A short 23% P700-saturating flash, having a duration of 3.5 μs at half-peak height, was used to avoid multiple turnovers of the cytochrome b6 fcomplex. Cytochrome f was monitored as ΔA554–545 nm (14Zhou J. Fernández-Velasco J.G. Malkin R. J. Biol. Chem. 1996; 271: 6225-6232Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) in the presence of 30 μm FCCP. For the measurement of cytochrome foxidation, 22 μm stigmatellin was also added to inhibit the reduction of cytochrome f. Cytochromeb6 was measured as ΔA564–575 nm (15Soriano G.M. Ponamarev M.V. Tae G.-S. Cramer W.A. Biochemistry. 1996; 35: 14590-14598Crossref PubMed Scopus (72) Google Scholar) in the presence of 30 μm HQNO and 30 μm FCCP. The slow phase of the electrochromic band shift was measured as the difference of ΔA515 nm (18Ponamarev M.V. Cramer W.A. Biochemistry. 1998; 37: 17199-17208Crossref PubMed Scopus (61) Google Scholar) in the absence and the presence of 22 μm stigmatellin. The kinetic data were fit as first order reactions to give the rate constants, which were used to calculate the half-times of the reactions. DNA was isolated from the primary transformants, which were able to grow on the TAP plates containing spectinomycin. The colonies giving the correct size of the DNA fragment by PCR were transferred to HS plates and incubated under dim light at 25 °C. Most of the colonies died after 3–4 weeks. Only a few tiny green colonies were found under the microscope. They were restreaked onto the fresh HS plates and TAP plates. After a few weeks it was confirmed that pFSSD mutant was able to grow photoautotrophically. The growth rate measured in HS liquid medium under 35 μmol of photons m−2 s−1 showed that the pFSSD mutant grew slightly more slowly than the wild type (TableI). Under higher light intensity,i.e. 160 μmol of photons m−2s−1, the slower growth in the pFSSD mutant is more noticeable (doubling time, 9.8 h versus 8 h). Southern blot analysis revealed the presence of the correct insertion in these cells (Fig. 3). In comparison with the wild type strain, the smaller band from the pFSSD mutant showed the correct size of deletion (∼0.16 kilobase) from thepetA gene sequence. The chlorophyll contents of the wild type and pFSSD mutant are very similar. The chlorophyll (a +b)/cell is 3.8 ± 0.5 × 10−15 and 3.2 ± 0.3 × 10−15 mol/cell, respectively. The chlorophyll a/chlorophyll b ratio is 2.5 and 2.3, respectively. The presence of the mutant polypeptide was then examined by immunoblotting (Fig. 4). The anti-cytochromef antibodies identified a band of smaller molecular mass (∼25 kDa) in the pFSSD mutant, which is approximately the expected molecular mass for cytochrome f after the deletion of the small domain. The antibodies against cytochromeb6, subunit IV, and Rieske FeS protein also identified bands of the same size as the wild type from the pFSSD mutant, which are in contrast to the petA deletion strain where no detection of any of the subunits of the cytochromeb6 f complex is observed (Fig. 4,lane 2). It is known that the absence of cytochromef results in a rapid degradation of the cytochromeb6 f complex subunits (3Kuras R. Wollman F.-A. EMBO J. 1994; 13: 1019-1027Crossref PubMed Scopus (155) Google Scholar). Therefore the presence of other subunits in the pFSSD mutant suggests that the cytochrome b6 f complex was assembled. However, the level of these proteins in the mutant cells was only 40–30% of the level in the wild type cells, showing that there was less cytochrome b6 f complex in the mutant. Detection of peroxidase activity using tetramethylbenzidine and H2O2 also indicated the presence of a new 25-kDa heme-containing protein, suggesting that heme had been covalently inserted into the mutant polypeptide of cytochromef. We conclude from these studies that deletion of the small domain does not affect the translocation of the protein to the lumen, the processing of the precursor protein, and the incorporation of the heme group into the polypeptide and that the small domain is not essential for the assembly of the cytochromeb6 f complex.Table IComparison of physiological and kinetic parameters between wild type and the pFSSD mutantDoubling time1-aCells were grown in the HS medium under 35 μmol of photons m−2s−1 and bubbled with 3% CO2.Oxygen evolution1-bNet photosynthesis. Incident intensity 2530 μmol of photons m−2s−1. Experiments were performed at 25 °C.t 12Cytochrome f oxidationCytochrome freductionCytochrome b6 reductionSlow Δψ 515 nmhnmol O2 μmol Chl−1min−1msWild type12.4 ± 0.3 (4)1-cThe number of total trials is given in parentheses for each case.2222 ± 81 (3)0.14 ± 0.03 (5)2.1 ± 0.3 (5)4.7 ± 0.4 (8)2.7 ± 0.2 (6)pFSSD13.9 ± 0.3 (4)400 ± 2 (2)0.10 ± 0.04 (8)32 ± 5 (10)8.6 ± 2.9 (9)5.5 ± 0.7 (6)The range represents the maximum variation of data collected from different experiments with different cultures.1-a Cells were grown in the HS medium under 35 μmol of photons m−2s−1 and bubbled with 3% CO2.1-b Net photosynthesis. Incident intensity 2530 μmol of photons m−2s−1. Experiments were performed at 25 °C.1-c The number of total trials is given in parentheses for each case. Open table in a new tab Figure 4Immunoblot analysis (A) and heme staining (B) of the cytochromeb6 f complex subunits from wild type and the pFSSD mutant of C. reinhardtii.Total cellular protein from C. reinhardtii wild type (lane 1), ΔpetA (lane 2), and pFSSD" @default.
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• W1986201304 title "Electron Transfer and Stability of the Cytochromeb6 f Complex in a Small Domain Deletion Mutant of Cytochrome f" @default.
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