Portal de Periódicos da CAPES

Ferredoxin:NADP+ Oxidoreductase Is a Subunit of the Chloroplast Cytochrome bfComplex

2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês

10.1074/jbc.m105454200

1083-351X

Huamin Zhang, Julian P. Whitelegge, William A. Cramer,

Redox biology and oxidative stress

Purified detergent-soluble cytochromeb6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300–350 electrons percyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f α-band and the Qy chlorophylla band in the cyanobacterial complex and an absorbance band in the flavin 450–480-nm region of the chloroplast complex. An additional high molecular weight (Mr ∼ 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of ∼0.9 (cytochrome f)−1. The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplastb6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport. Purified detergent-soluble cytochromeb6f complex from chloroplast thylakoid membranes (spinach) and cyanobacteria (Mastigocladus laminosus) was highly active, transferring 300–350 electrons percyt f/s. Visible absorbance spectra showed a red shift of the cytochrome f α-band and the Qy chlorophylla band in the cyanobacterial complex and an absorbance band in the flavin 450–480-nm region of the chloroplast complex. An additional high molecular weight (Mr ∼ 35,000) polypeptide in the chloroplast complex was seen in SDS-polyacrylamide gel electrophoresis at a stoichiometry of ∼0.9 (cytochrome f)−1. The extra polypeptide did not stain for heme and was much more accessible to protease than cytochrome f. Electrospray ionization mass spectrometry of CNBr fragments of the 35-kDa polypeptide was diagnostic for ferredoxin:NADP+ oxidoreductase (FNR), as were antibody reactivity to FNR and diaphorase activity. The absence of FNR in the cyanobacterial complex did not impair decyl-plastoquinol-ferricyanide activity. The activity of the FNR in the chloroplastb6f complex was also shown by NADPH reduction, in the presence of added ferredoxin, of 0.8 heme equivalents of the cytochrome b6 subunit. It was inferred that the b6f complex with bound FNR, one equivalent per monomer, provides the membrane protein connection to the main electron transfer chain for ferredoxin-dependent cyclic electron transport. iron-sulfur protein ferredoxin-NADPH+ oxidoreductase polyacrylamide gel electrophoresis photosystem n-undecyl-β-d-maltopyranoside The cytochrome b6f complex provides the electronic connection between the two reaction center complexes of oxygenic photosynthesis and, by oxidizing the lipophilic plastoquinol and transferring the resulting protons to the electrochemically positive side of the membrane, also contributes significantly to the generation of the trans-membrane proton electrochemical potential (1Cramer W.A. Soriano G.M. Ponomarev M. Huang D. Zhang H. Martinez S.E. Smith J.L. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 477-508Crossref PubMed Scopus (163) Google Scholar). The complex is known to contain three redox-active polypeptide subunits, cytochrome f, cytochrome b6, and the Rieske iron-sulfur protein (ISP).1 Our understanding of the structure and function of this complex, and its relation to the cytochrome bc1 complex in the electron transport chain of mitochondrial respiration and photosynthetic bacteria (most recent and highest resolution structure; Ref. 2Hunte C. Koepke J. Lange C. Rossmanith T. Michel H. Structure. 2000; 8: 669-684Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar), has been extended in recent years: (i) high resolution structures have been obtained of the lumen-side soluble domain of cytochrome f in plant (3Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 4Martinez S. Huang D. Ponomarev M. Cramer W.A. Smith J.L. Protein Sci. 1996; 5: 1081-1092Crossref PubMed Scopus (132) Google Scholar), cyanobacterial (5Carrell 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), and algal (6Sainz G. Carrell C.J. Ponamarev M.V. Soriano G.M. Cramer W.A. Smith J.L. Biochemistry. 2000; 39: 9164-9173Crossref PubMed Scopus (65) Google Scholar, 7Chi Y.I. Huang L.S. Zhang Z. Fernandez-Velasco J.G. Berry E.A. Biochemistry. 2000; 39: 7689-7701Crossref PubMed Scopus (45) Google Scholar) sources and of the Rieske iron-sulfur protein from plants (8Zhang H. Carrell C.J. Huang H. Sled V. Ohnishi T. Smith J.L. Cramer W.A. J. Biol. Chem. 1996; 271: 31336-31360Google Scholar, 9Carrell C.J. Zhang H. Cramer W.A. Smith J.L. Structure. 1997; 5: 1613-1625Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar); (ii) the existence of small (∼4-kDa) hydrophobic polypeptides in the complex has been recognized (10Haley J. Bogorad L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1534-1538Crossref PubMed Scopus (60) Google Scholar); and (iii) a bound molecule of chlorophyll a (11Bald D. Kruip J. Boekema E.J. Roegner M. Murata N. Research in Photosynthesis: Proceedings of the Ninth International Congress on Photosynthesis. Kluwer Academic Publishers, Dordrecht1992: 629-632Crossref Google Scholar, 12Huang D. Everly R.M. Cheng R.H. Heymann J.B. Schägger H. Sled V. Ohnishi T. Baker T.S. Cramer W.A. Biochemistry. 1994; 33: 4401-4409Crossref PubMed Scopus (104) Google Scholar, 13Pierre Y. Breyton C. Kramer D. Popot J.L. J. Biol. Chem. 1995; 270: 29342-29349Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) and of carotenoid (14Zhang H. Huang D. Cramer W.A. J. Biol. Chem. 1999; 274: 1581-1587Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), apparently not found in the bacterialbc1 complex, has been recognized. Two-dimensional crystals of the b6f complex from Chlamydomonas reinhardtii have been obtained that provide projection maps to 8–9 Å (15Mosser G. Breyton C. Olofsson A. Popot J.-L. Rigoud J.-L. J. Biol. Chem. 1997; 272: 20263-20268Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 16Breyton C. J. Biol. Chem. 2000; 275: 13195-13201Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), and three-dimensional crystals of the complex from the thermophilic cyanobacterium Mastigocladus laminosusdiffract to somewhat lower resolution (17Huang D. Zhang H. Soriano G.M. Dahms T.E.S. Krahn J.M. Smith J.L. Cramer W.A. Photosynthesis: Mechanisms and Effects. 3. Kluwer Academic Publishers, Dordrecht1999: 1577-1580Google Scholar). As part of an effort to improve the quality of the three-dimensional crystals, the characterization of the b6f complex isolated from spinach thylakoids and M. laminosus has been extended. The present study documents that a fourth redox-active subunit in addition to cytochromes f and b6 and the Rieske ISP, ferredoxin:NADP+ oxidoreductase (FNR, 314 residues; mass, 35,315), is present in the cytochromeb6f complex isolated from spinach thylakoids but not in that from M. laminosus purified by the same protocol. The FNR bound stoichiometrically to the purifiedb6f complex is enzymatically active, implying a role of the b6f complex in the photosystem I (PSI)- and ferredoxin-dependent cyclic electron transport pathway. Thylakoid membranes were isolated as described by Hurt and Hauska (18Hurt E.C. Hauska G. Eur. J. Biochem. 1981; 117: 591-599Crossref PubMed Scopus (299) Google Scholar). Cytochromeb6f complex was extracted in TMKNE (30 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 5 mm KCl, 50 mm NaCl, and 1 mm EDTA) containing 0.2% sodium cholate and 28 mmn-octyl-β-d-glucoside at a chlorophyll concentration of 2 mg/ml. The chloroplast suspension was stirred at room temperature for 20 min and centrifuged at 300,000 ×g for 40 min, the supernatant was collected, and solid ammonium sulfate was added to 35% saturation. The precipitate was removed by centrifugation at 160,000 × g for 30 min, and the supernatant was loaded on a propyl-agarose column (1.5 × 10 cm) equilibrated with 35% saturated ammonium sulfate in TMKNE containing 0.05%n-undecyl-β-d-maltopyranoside (UDM). The column was washed with equilibration solution, and the cytochromeb6f complex was eluted with 10% saturated ammonium sulfate in TMKNE with 0.05% UDM. Fractions containing cytochrome b6f complex were pooled, concentrated in a Centriprep 10, loaded on a sucrose gradient (8–35%) in TMKNE and 0.05% UDM, and centrifuged at 35,000 rpm (16 h) in an SW-41 rotor. The brown band in the middle of the gradient was collected. Cells were harvested by centrifugation at 5000 × g (10 min) and resuspended in 25 mmHepes-KOH, pH 7.5, 10 mm CaCl2, 10 mm MgCl2, 0.4 m sucrose, 0.25 mm phenylmethylsulfonyl fluoride, 2 mmbenzamidine, and 2 mm ε-amino-caproic acid. The cell suspension was passed through a French pressure cell at 18,000 pounds/square inch. Unbroken cells were removed by centrifugation at 3000 × g (10 min), and thylakoid membranes were collected by centrifugation at 90,000 × g (45 min). The sediment was resuspended in 30 mm Tricine, pH 8.0, and washed according to the procedure described for spinach thylakoid membranes, and cytochrome b6f complex was extracted and purified as described above for the spinach complex. Plastocyanin was purified according to the procedure of Morand and Krogmann (19Morand L.Z. Krogmann D.W. Biochim. Biophys. Acta. 1993; 1141: 105-106Crossref Scopus (12) Google Scholar). Solid ammonium sulfate was added to the supernatant of broken M. laminosus cells to achieve 60% saturation. The precipitate was removed by centrifugation at 35,000 × g (30 min), the supernatant was collected, and ammonium sulfate was added to 90% saturation. The precipitate containing plastocyanin was sedimented by centrifugation at 36,000 × g (30 min), and the pellet was resuspended in 50 mm potassium phosphate, pH 7.0, and 1 mmferricyanide and dialyzed against 5 mm potassium phosphatei, pH 7.0, overnight. This fraction was loaded onto a diethylaminoethyl-cellulose column equilibrated with 10 mm potassium phosphatei, pH 7.0. The void fraction containing plastocyanin was collected and concentrated. The concentrated plastocyanin fraction was subsequently loaded on a CM-Sepharose column equilibrated with 10 mm Tris-HCl, pH 8.0. Pure plastocyanin was eluted in 10 mm Tris-HCl, pH 8.0, and 50 mm NaCl. Chemical difference spectra of cytochromes f andb6 and plastocyanin were measured using a Cary 3 UV-visible spectrophotometer with a measuring beam half-bandwidth of 2 nm. Plastocyanin oxidoreductase activity of cytochromeb6f complex was assayed on a Aminco-Chance dual-beam spectrophotometer. The assay mixture contained 125 μm ferricyanide, 5 μm plastocyanin (from spinach or M. laminosus), 5 nm cytochromeb6f in 30 mm4-morpholino-ethanesulfonic acid, pH 6.0, 50 mm NaCl, and 2 mm EDTA. Reduction of plastocyanin, initiated by addition of 25 μm decyl-plastoquinol (DPQH2), was monitored as the absorbance change at 600 nm relative to 500 nm based on an extinction coefficient of 4.9 mm−1(20Katoh S. Shiratori I. Takamiya A. J. Biochem. Tokyo. 1962; 51: 32-40Crossref PubMed Scopus (222) Google Scholar). Diaphorase activity of bound FNR was measured according to the method of Avron and Jagendorf (21Avron D.I. Jagendorf A.T. Arch. Biochem. Biophys. 1956; 65: 475-483Crossref PubMed Scopus (108) Google Scholar). The reaction mixture contained 30 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm EDTA, 0.05% UDM, 35 nm cytochromeb6f complex, and 20 μm2,6-dichloro-indophenol. The reduction of 2,6-dichloro-indophenol by NADPH (67 μm) was measured by the absorbance change at 620 nm relative to 500 nm and the rate of reduction of 2,6-dichloro-indophenol based on an oxidized minus reduced millimolar extinction coefficient of 20. SDS-polyacrylamide gel electrophoresis (PAGE) was performed on an Amersham Pharmacia Biotech Phast gel system with constant temperature control using precast gels containing 20% acrylamide. Samples were prepared in 50 mm Tris-HCl, pH 8.0, 4m urea, 2% SDS, 5% glycerol, and 2.5% mercaptoethanol and heated for 2 min at 90 °C before loading. Blue native gel electrophoresis was carried out by the method described by Shägger and von Jagow (22Schägger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1901) Google Scholar) and Schägger et al. (23Schägger H. Cramer W.A. von Jagow G. Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1037) Google Scholar); a 1-mm thick 8–16% gradient gel was used. Cytochromeb6f was dissolved in 25 mmBis-Tris, pH 7.0, 0.15% UDM, and 0.05% Serva Blue G. Heme-containing proteins on SDS-PAGE were stained using 3,3′,5,5′-tetramethyl-benzidine according to the method of Thomas et al (24Thomas P.E. Ryan D. Levin W. Anal. Biochem. 1976; 75: 168-176Crossref PubMed Scopus (896) Google Scholar). Proteins were electrotransferred from gel slabs used in SDS-PAGE to polyvinylidene difluoride membranes for 1 h at 130 mA in a Hoefer TE70 semidry transfer blotting system. Immunoblotting used horseradish peroxidase-conjugated IgG at 1:2000 dilution. For the color reaction, the polyvinylidene difluoride membrane was soaked in 10 mm Tris-HCl, pH 7.5, and 150 mm NaCl containing 0.015% chloronaphthol, which was diluted from a freshly made 0.3% solution in ethanol. After 5 min of incubation, 0.2% H2O2 was added. The reaction was terminated by rinsing the membrane with water. Cytochrome b6f complex from spinach chloroplasts was treated with thermolysin at a ratio of 1:100 (w/w) at room temperature for 2 h. Thermolysin and proteolytically cleaved polypeptides were removed using a Centricon 100 centrifuge at 1000 × g. The results of the proteolysis were assayed by SDS-PAGE. M. laminosus genomic DNA was purified according to the method of Ausubel et al (25Current Protocols in Molecular Biology, Preparation and Analysis of DNA. Vol. 1. John Wiley and Sons, New York2000Google Scholar). The petA gene was amplified by PCR using degenerate primer pairs based on thepetA gene sequence from Nostoc. Restriction sites (BamHI and EcoRI) for cloning were incorporated into the PCR primers. The DNA fragments obtained from PCR were purified from an agarose gel, digested with BamHI andEcoRI, and cloned in pBluescript II KS for sequence determination. Samples (100 μg of protein) were precipitated with chloroform/methanol and dissolved in 60% formic acid before immediate size exclusion chromatography-electrospray ionization mass spectrometry (Tosohaas Super SW2000, 4.6 × 300 mm, 40 °C) in chloroform/methanol/1% aqueous formic acid (4:4:1, v/v) as described previously (26Whitelegge J.P. le Coutre J. Lee J.C. Engel C.K. Prive G.G. Faull K.F. Kaback H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10695-10698Crossref PubMed Scopus (106) Google Scholar, 27le Coutre J. Whitelegge J.P. Gross A. Turk E. Wright E.M. Kaback H.R. Faull K.F. Biochemistry. 2000; 39: 4237-4242Crossref PubMed Scopus (90) Google Scholar). Spectra were recorded with an API III+ quadrupole instrument equipped with an Ionspray TM source (PerkinElmer Sciex), tuned and calibrated as previously described (28Whitelegge J.P. Gundersen C. Faull K.F. Protein Sci. 1998; 7: 1423-1430Crossref PubMed Scopus (167) Google Scholar). Mass spectra from the appropriate part of the chromatogram were deconvoluted to the zero-charge molecular weight spectrum (Bio Multiview, 1.3.1; Perkin Elmer Sciex) as described elsewhere. 2J. P. Whitelegge, H. Zhang, and W. A. Cramer, manuscript in preparation. Purified cytochrome b6fcomplex from spinach thylakoids and the cyanobacterium M. laminosus showed similar absorbance spectra except for: (i) a 2-nm red shift (554–556 nm) of the reduced cytochrome f α-band (Fig. 1A, peak 4) caused by the residue shift Phe4→Trp4, as described by Ponamarev et al. (30Ponamarev M.V. Schlarb B.G. Howe C.J. Carrell C.J. Smith J.L. Bendall D.S. Cramer W.A. Biochemistry. 2000; 39: 5971-5976Crossref PubMed Scopus (29) Google Scholar); (ii) a 3–4-nm red shift (from 668–669 to 672 nm) in the chlorophyll a Qy band (Fig. 1A, peak 5) in the M. laminosus spectrum (14Zhang H. Huang D. Cramer W.A. J. Biol. Chem. 1999; 274: 1581-1587Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar); and (iii) a small peak in the 450–480-nm region (Fig. 1A, peak 2) that could be characteristic of the FAD (31Batie C.J. Kamin H. J. Biol. Chem. 1981; 256: 7756-7763Abstract Full Text PDF PubMed Google Scholar). When chloroplast (Fig. 1B, solid line) and M. laminosus (Fig. 1B, dashed line) difference spectra (dithionite minus ascorbate in the presence of glucose oxidase) are amplified and compared in the 400–500-nm spectral region, it is apparent that the spectral peak of the chloroplast b6f complex in the 450–480-nm region is missing from the M. laminosusdifference spectrum (Fig. 1B, arrow). Using an extinction coefficient at 480 nm, εmM = 7.4 (31Batie C.J. Kamin H. J. Biol. Chem. 1981; 256: 7756-7763Abstract Full Text PDF PubMed Google Scholar), the amplitude of this absorbance band implies a FAD content of 0.8–1.0 per cytochromef. The 450–480-nm difference spectra of the chloroplast b6f complex resemble reduced minus oxidized spectra of FNR generated by flash illumination ofChlorella (32Bouges-Bocquet B. FEBS Lett. 1978; 85: 340-344Crossref PubMed Scopus (18) Google Scholar). A comparison of SDS-PAGE profiles of the large (>15-kDa) subunits of the cytochrome b6fcomplex from spinach thylakoids (Fig. 2,lane 2) and the thermophilic cyanobacterium M. laminosus (Fig. 2, lane 3) shows the presence of the four well known polypeptides in the b6fcomplex. In order of descending molecular size, these are cytochromef, cytochrome b6, the Rieske iron-sulfur protein, and subunit IV, with Mr values of 33,000, 24,000, 21,000, and 18,000, respectively. In this gel system (20% acrylamide), there is an overlap between theb6 and Rieske bands in the Mr21,000–24,000 region of the spinach complex, as seen in the high staining density of this band (Fig. 2, lane 2). These subunits are separated in the M. laminosus complex (Fig. 2,lane 3). There is no doubt from the difference spectra (Fig.1B), heme stain (Fig. 3), electron transfer activity, and mass spectrometry of the intact complex2 that the spinach complex contains a full component of the cytochrome b6 and Rieske ISP subunits.Figure 3Heme-stained profile of SDS-PAGE of spinach cytochrome b6fcomplex. Lane 1, molecular weight standards; lane 2, spinach cytochrome b6f complex;lane 3, 3,3′,5,5′-tetramethyl-benzidine stain. Conditions were as described under "Materials and Methods."View Large Image Figure ViewerDownload Hi-res image Download (PPT) The difference in mobility of cytochrome f is attributed to differences in amino acid sequences of the two cytochromes f(Table I). There is 58% identity between the two cytochromes f, with a similar content of hydrophobic residues, 53.6 and 54.7%, respectively. The basis for the mobility difference of the Rieske protein is not known, because its nucleotide sequence in M. laminosus has not been determined.Table ISequence alignment of cytochrome f (pet A) from spinach and M. laminosusAlignment was obtained using Clustal W (54Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55621) Google Scholar). *, identical or conserved residues; ∶, conserved substitutions; ., semiconserved substitutions. Open table in a new tab Alignment was obtained using Clustal W (54Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55621) Google Scholar). *, identical or conserved residues; ∶, conserved substitutions; ., semiconserved substitutions. In addition, a fifth polypeptide with the largest Mr value, 35,000, of the subunit polypeptides is seen above the Mr∼33,000 cytochrome f band from chloroplasts (Fig. 2,lane 2) but not in cyanobacteria (Fig. 2, lane 3). The presence of the fifth polypeptide in the chloroplast and not the cyanobacterial complex is reflected in a smallerMr value of the M. laminosus compared with the spinach complex measured on the native gel system of Schägger et al. (22, 23; data not shown). TheMr 35,000 polypeptide appears to be bound stoichiometrically to the other large subunits of the complex, as seen in Fig. 2. Assuming that the binding of Coomassie blue stain is proportional to molecular weight, the densitometric ratio of the subunits in the SDS-PAGE of Fig. 2, normalized relative to cytochromef, is 0.95 (35-kDa subunit):1.0 (cyt f):1.05 (b6 and Rieske ISP):0.85 (subunit IV) (average of two sets of scans). The observation of the fifth polypeptide in the complex was a major aspect of the original study by Hurt and Hauska (18Hurt E.C. Hauska G. Eur. J. Biochem. 1981; 117: 591-599Crossref PubMed Scopus (299) Google Scholar) on the purification and properties of the cytochromeb6f complex from spinach. Their study did not detect a difference in the heme-staining properties of the two bands in the Mr 33,000–34,000 cytochromef region. They suggested that the two bands in this region arose from cytochromes f and a polypeptide containing one of the two hemes of cytochrome b6, respectively, because it was not known at this time that the two hemes of cytochromeb6 are bound to the same 23-kDa polypeptide. The two bands in the cytochrome f region were also noted by Romanowska and Albertsson (33Romanowska E. Albertsson P.-A. Plant Cell Physiol. 1994; 35: 557-568Crossref Scopus (21) Google Scholar), who did not resolve a difference in their heme-staining properties and concluded that both bands belonged to forms of cytochrome f. However, it was found in the present study that the fifth and largest (Mr35,000) polypeptide in the SDS-PAGE of the spinach chloroplast complex did not stain for heme (Fig. 3). This was observed under conditions in which the second and third largest (Mr 33,000 and 24,000) polypeptides in the spinach complex, those associated with cytochromes f and b6 in the four-component gel of M. laminosus, did react with the heme-staining reagent (Fig. 3). A different degree of integration into the spinach complex of theMr 35,000 and 33,000 polypeptides is implied by the complete accessibility and susceptibility of theMr 35,000 but not the Mr33,000 polypeptide, to the protease thermolysin (Fig.4, lane 5 versus lanes 2–4). Cytochrome f in the thylakoid membrane is known to be inaccessible to a range of proteases (34Szczepaniak A. Black M.T. Cramer W.A. Z. Naturforsch. 1989; 44c: 453-461Crossref Scopus (14) Google Scholar). It can be seen that the Rieske ISP and subunit IV polypeptides are also sensitive to the protease (Fig. 4, lane 5). Although theMr 35,000 component is accessible to protease, implying a peripheral location in the complex, it cannot be removed by washing with high concentrations of NaBr or EDTA (Fig. 4, lanes 3 and 4 versus lane 2). The absence of heme staining of the Mr 35,000 polypeptide in the spinach complex and the similarity of its Mr value to that of the "true" Mr 33,000 spinach cytochrome f suggested that the Mr35,000 polypeptide might be preapocytochrome, i.e.cytochrome f with its 35-residue leader peptide (35Alt J. Herrmann R.G. Curr. Genet. 1984; 8: 551-557Crossref PubMed Scopus (87) Google Scholar) but without the heme, the incorporation of which requires a free N terminus of the mature cytochrome f (3Martinez S.E. Huang D. Szczepaniak A. Cramer W.A. Smith J.L. Structure. 1994; 2: 95-105Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Electrospray mass ionization mass spectroscopy displayed a component in the mass spectrum of the spinach b6f complex with a mass of 35,314, greater than the mass component of 31,972 attributed to mature holocytochrome f (Fig.5A). The 34,624 mass component is an artifact with a mass that corresponds to that of a dimer of subunit IV. The 35-kDa component was absent from the mass spectrum of the b6f complex from M. laminosus (Fig. 5B). The 35,314 mass of the high molecular weight component in the spinachb6f complex was indeed almost the same as that (Mr 35,328) calculated for acetylated preapocytochrome f but as well was very similar to that of FNR (Mr 35,315). Western blots of theMr 35,000 polypeptide in the spinach complex showed that it reacted with an antibody to spinach FNR (Fig.6, lane 4 versus lane 2), whereas the Mr 33,000 polypeptide reacted with an antibody to cytochrome f (Fig.6, lane 3 versus lane 2). Furthermore, electrospray ionization mass spectroscopy of CNBr fragments of theMr 35,314 polypeptide, isolated chromatographically from the chloroplast complex, ranging from 3020 to 6538 in molecular weight, were similar to those expected from FNR but not from cytochrome f (TableII). The Mr 1145 and 5657 CNBr fragments (Table II, first column) are thought to result from nonspecific cleavage.Figure 6Western blot with antibody to cytochromef (lane 3) and FNR (lane 4) of spinach cytochrome b6f complex. Lane 1, molecular weight standards; lane 2, spinach cytochrome b6f complex; lanes 3 and 4, immune blots with antibody to cytochromef and FNR, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIComparison of intact spinach 35-kDa polypeptide and its CNB fragments with calculated masses of FNR and cytochrome f by electrospray ionization mass spectroscopyMeasured mass of 35-kDa polypeptide2-aIntact masses were measured on API III (Sciex) and computed from raw spectra (Hypermass; Sciex); fragments on LCQ-DECA (Thermoquest) with Turbosequest software (Thermoquest). The intact mass reported here is 6 mass units higher than the value obtained with the software (BioMultiView 1.3.1, Sciex). See Fig. 5. and CNBr fragmentsCalculated mass of FNR2-bMasses were calculated from SwissProt entries P00455 and P16013 with modifications using PeptideMass (www.expasy.org) set to generate the average mass for the uncharged species based on natural isotopic abundance. Only FNR fragments with sizes similar to the observed masses are shown.2-cFNR with Q1 of mature form; pre apocyt f with initiating Met removed and N-acetylated in calculation. and fragments2-bMasses were calculated from SwissProt entries P00455 and P16013 with modifications using PeptideMass (www.expasy.org) set to generate the average mass for the uncharged species based on natural isotopic abundance. Only FNR fragments with sizes similar to the observed masses are shown.Calculated mass of preapocyt f2-bMasses were calculated from SwissProt entries P00455 and P16013 with modifications using PeptideMass (www.expasy.org) set to generate the average mass for the uncharged species based on natural isotopic abundance. Only FNR fragments with sizes similar to the observed masses are shown.2-cFNR with Q1 of mature form; pre apocyt f with initiating Met removed and N-acetylated in calculation. and fragments2-bMasses were calculated from SwissProt entries P00455 and P16013 with modifications using PeptideMass (www.expasy.org) set to generate the average mass for the uncharged species based on natural isotopic abundance. Only FNR fragments with sizes similar to the observed masses are shown.DaDa35,320.3 ± 5.935,314 (1–313)2-dSubtracted 17 for N-terminal pyroGlu.2-eSubtracted 2 for single disulfide; assumed F268V (29).35,328.7 (2–318)CNBr1,145.4 ± 0.2486.6 (129–132)3,020.2 ± 0.73,020.3 (220–245)4,097.7 ± 0.74,099.6 (24–60)3,952.6 (91–128)4,357.9 ± 0.54,355.9 (278–314)2-fAdded 16 because of single internal MetO, which resulted in a missed cleavage.4,485.3 (91–132)2-fAdded 16 because of single internal MetO, which resulted in a missed cleavage.5,657.2 ± 0.76,538.4 ± 1.26,539.5 (1–60)2-dSubtracted 17 for N-terminal pyroGlu.2-fAdded 16 because of single internal MetO, which resulted in a missed cleavage.10,018.6 (2–90)20,471.5 (133–318)2-a Intact masses were measured on API III (Sciex) and computed from raw spectra (Hypermass; Sciex); fragments on LCQ-DECA (Thermoquest) with Turbosequest software (Thermoquest). The intact mass reported here is 6 mass units higher than the value obtained with the software (BioMultiView 1.3.1, Sciex). See Fig. 5.2-b Masses were calculated from SwissProt entries P00455 and P16013 with modifications using PeptideMass (www.expasy.org) set to generate the average mass for the uncharged species based on natural isotopic abundance. Only FNR fragments with sizes similar to the observed masses are shown.2-c FNR with Q1 of mature form; pre apocyt f with initiating Met removed and N-acetylated in calculation.2-d Subtracted 17 for N-terminal pyroGlu.2-e Subtracted 2 for single disulfide; assumed F268V (29Karplus A.P. Walsh K.A. Herriott J.R. Biochemistry. 1984; 23: 6576-6583Crossref PubMed Scopus (88) Google Scholar).2-f Added 16 because of single internal MetO, which resulted in a missed cleavage. Open table in a new tab Diaphorase activity (5–10 electrons per cyt f/s), arising from the presence of FNR, was readily detected in the spinach b6f complex (Fig.7, trace 1), identical to that in an EDTA-washed complex