The Interactions of Cyanobacterial Cytochromec6 and Cytochrome f, Characterized by NMR
2002; Elsevier BV; Volume: 277; Issue: 50 Linguagem: Inglês
10.1074/jbc.m203983200
ISSN1083-351X
AutoresPeter B. Crowley, Antonio Díaz‐Quintana, Fernando P. Molina-Heredia, Pedro M. Nieto, M. Sutter, Wolfgang Haehnel, Miguel Á. De la Rosa, Marcellus Ubbink,
Tópico(s)Enzyme Structure and Function
ResumoDuring oxygenic photosynthesis, cytochromec6 shuttles electrons between the membrane-bound complexes cytochrome bf and photosystem I. Complex formation between Phormidium laminosum cytochromef and cytochrome c6 from bothAnabaena sp. PCC 7119 and Synechococcus elongatus has been investigated by nuclear magnetic resonance spectroscopy. Chemical-shift perturbation analysis reveals a binding site on Anabaena cytochrome c6, which consists of a predominantly hydrophobic patch surrounding the heme substituent, methyl 5. This region of the protein was implicated previously in the formation of the reactive complex with photosytem I. In contrast to the results obtained for Anabaena cytochromec6, there is no evidence for specific complex formation with the acidic cytochrome c6 fromSynechococcus. This remarkable variability between analogous cytochromes c6 supports the idea that different organisms utilize distinct mechanisms of photosynthetic intermolecular electron transfer. During oxygenic photosynthesis, cytochromec6 shuttles electrons between the membrane-bound complexes cytochrome bf and photosystem I. Complex formation between Phormidium laminosum cytochromef and cytochrome c6 from bothAnabaena sp. PCC 7119 and Synechococcus elongatus has been investigated by nuclear magnetic resonance spectroscopy. Chemical-shift perturbation analysis reveals a binding site on Anabaena cytochrome c6, which consists of a predominantly hydrophobic patch surrounding the heme substituent, methyl 5. This region of the protein was implicated previously in the formation of the reactive complex with photosytem I. In contrast to the results obtained for Anabaena cytochromec6, there is no evidence for specific complex formation with the acidic cytochrome c6 fromSynechococcus. This remarkable variability between analogous cytochromes c6 supports the idea that different organisms utilize distinct mechanisms of photosynthetic intermolecular electron transfer. Electron transport between the membrane-bound complexes cytochromebf and photosystem I (PSI) 1The abbreviations used are: PSI, photosystem I; Pc, plastocyanin; cytc , cytochromec ; cytc6, cytochromec6; cytf , cytochrome f ; Ana , Anabaena; Syn , Synechococcus; HSQC, heteronuclear single quantum correlation spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy. is maintained by mobile electron carriers. In plants, this task is fulfilled by plastocyanin (Pc), whereas cytochrome c6(cytc6) is the only carrier in certain cyanobacteria. There also exist eukaryotic algae and cyanobacteria, which have the capacity to replace Pc with cytc6under copper-depleted conditions (1Wood P.M. Eur. J. Biochem. 1978; 87: 9-19Google Scholar). Recently, a cytc6-like protein has been discovered in the thylakoid lumen of Arabidopsis (2Wastl J. Bendall D.S. Howe C.J. Trends Plant Sci. 2002; 7: 244-245Google Scholar, 3Gupta R. He Z.Y. Luan S. Nature. 2002; 417: 567-571Google Scholar). Despite being evolutionarily unrelated, Pc and cytc6 perform equivalent tasks with common reaction partners. This functional convergence suggests that similar interaction properties exist for both proteins. Not surprisingly, Pc and cytc6 have comparable redox potentials of around 350 mV. Furthermore, both proteins provide similar recognition information, demonstrated by the parallel variation of their isoelectric points, being acidic in green algae while ranging from acidic to basic in cyanobacteria (1Wood P.M. Eur. J. Biochem. 1978; 87: 9-19Google Scholar, 4Navarro J.A. Hervás M. De la Rosa M.A. J. Biol. Inorg. Chem. 1997; 2: 11-22Google Scholar,5Kerfeld C.A. Krogmann D.W. Annu. Rev. Plant Physiol. 1998; 49: 397-425Google Scholar). To date there has been a considerable amount of kinetic and mutational analysis of the electron transfer reaction between both Pc and cytc6 and their partner PSI (6Hervás M. Navarro J.A. Dı́az A. Bottin H. De la Rosa M.A. Biochemistry. 1995; 34: 11321-11326Google Scholar, 7Hervás M. Navarro J.A. Dı́az A. De la Rosa M.A. Biochemistry. 1996; 35: 2693-2698Google Scholar, 8Molina-Heredia F.P. Dı́az-Quintana A. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 1999; 274: 33565-33570Google Scholar, 9Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 2001; 276: 601-605Google Scholar, 10Hippler M. Drepper F. Haehnel W. Rochaix J.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7339-7344Google Scholar, 11Illerhaus J. Altschmied L. Reichert J. Zak E. Herrmann R.G. Haehnel W. J. Biol. Chem. 2000; 275: 17590-17595Google Scholar). A hierarchy of mechanisms for interprotein electron transport has emerged from this work. Reduction of PSI by Pc or cytc6, isolated from different organisms, can follow an oriented collision mechanism (type I), a two-step mechanism requiring complex formation (type II), or a complex formation with rearrangement of the interface before electron transfer occurs (type III). A remarkable homology between the arrangement of charged and hydrophobic recognition patches on the surfaces of Pc and cytc6 has also been revealed (12Frazão C. Soares C.M. Carrondo M.A. Pohl E. Dauter Z. Wilson K.S. Hervas M. Navarro J.A. De la Rosa M.A. Sheldrick G.M. Structure. 1995; 3: 1159-1169Google Scholar, 13Ullmann G.M. Hauswald M. Jensen A. Kostic N.M. Knapp E.W. Biochemistry. 1997; 36: 16187-16196Google Scholar). In particular, a conserved arginine found in cyanobacterial Pc and cytc6 was shown to be critical for bimolecular association with PSI (9Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 2001; 276: 601-605Google Scholar). Two structures of the cytf-Pc complex from plant (14Ubbink M. Ejdeback M. Karlsson B.G. Bendall D.S. Structure. 1998; 6: 323-335Google Scholar) and cyanobacterial (15Crowley P.B. Otting G. Schlarb-Ridley B.G. Canters G.W. Ubbink M. J. Am. Chem. Soc. 2001; 123: 10444-10453Google Scholar) sources have been determined. A combination of charged and hydrophobic patches defines the complex interface between turnip cytf and spinach Pc. In contrast, the complex from Phormidium laminosum, a thermophilic cyanobacterium, was found to be predominantly hydrophobic. At present there are no kinetic or structural data for the interaction of cytc6 and cytf. Although mutagenesis studies have identified key residues for the reaction with PSI, there is no knowledge of the interaction site involved with cytf. To address the question of molecular recognition in cytc6, we have investigated complex formation with cytf using heteronuclear NMR. We have aimed to identify the surface features involved in complex formation and to draw comparisons with cytc6-PSI interactions. Two cyanobacterial variants of cytc6 (Fig. 1), the basic protein fromAnabaena sp. PCC 7119 (pI 9.0) and the acidic protein fromSynechococcus elongatus (pI 4.8), have been studied. In this way, the role of electrostatics in protein interactions could be investigated explicitly. Cytf from P. laminosum, with a net charge of −14, was used as the partner protein. It is important to note that P. laminosum cytf shares 74 and 72% sequence identity with Anabaena cytfand S. elongatus cytf, respectively. Furthermore, the net charge is intermediate of cytf fromAnabaena (−16) and S. elongatus (−12). This study reports the first structural characterization of the interactions of cytf and cytc6. The soluble fragment of cytfwas prepared according to previously published methods (15Crowley P.B. Otting G. Schlarb-Ridley B.G. Canters G.W. Ubbink M. J. Am. Chem. Soc. 2001; 123: 10444-10453Google Scholar, 16Schlarb B.G. Wagner M.J. Vijgenboom E. Ubbink M. Bendall D.S. Howe C.J. Gene (Amst.). 1999; 234: 275-283Google Scholar). Unlabeled Ana-cytc6 was produced inEscherichia coli GM119 (17Marinus M.G. Morris N.R. J. Bacteriol. 1973; 14: 1143-1150Google Scholar) transformed with both pEAC-WT (18Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. Biochem. Biophys. Res. Commun. 1998; 243: 302-306Google Scholar) and pEC86 (19Arslan E. Schulz H. Zufferey R. Künzaler P. Thöny-Meyer L. Biochem. Biophys. Res. Commun. 1998; 251: 744-747Google Scholar). The culture was grown in LB medium (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) supplemented with 100 μg/ml ampicillin, 10 μg/ml chloramphenicol, and 1 mm FeCl3. Aerobic growth was maintained at 37 °C for 18 h before harvesting. A similar expression system in E. coli JM109 was used for the production of uniformly 15N-labeledAna-cytc6. The culture was grown in M9 minimal media additionally supplemented with 1 g/liter15NH4Cl and 1 mm thiamine. Aerobic growth was maintained at 37 °C for 72 h before harvesting. Isolation and purification ofAna-cytc6 were achieved as described previously (8Molina-Heredia F.P. Dı́az-Quintana A. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 1999; 274: 33565-33570Google Scholar, 18Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. Biochem. Biophys. Res. Commun. 1998; 243: 302-306Google Scholar). The preparation and purification of15N, 13C double-labeledSyn-cytc6 have been reported previously (21Sutter M. Sticht H. Schmid R. Hörth P. Rösch P. Haehnel W. Mathis P. Photosynthesis: From Light to Biosphere. II. Kluwer Academic Publishers, The Netherlands1995: 563-566Google Scholar). Protein interactions were investigated for the diamagnetic species only, and reducing conditions were maintained in the presence of sodium ascorbate. Protein solutions were concentrated to the required volume using ultrafiltration methods (Amicon; YM3 membrane) and exchanged into 10 mm potassium phosphate, pH 6.0, 10% D2O, 1.0 mm sodium ascorbate. Protein concentrations were determined spectrophotometrically using an ε553 of 26.2 mm−1cm−1 for the ferrous form of both Ana-cytc6 (22Medina M. Louro R.O. Gagnon L. Peleato M.L. Mendes J. Gómez-Moreno C. Xavier A.V. Teixeira M. J. Biol. Inorg. Chem. 1997; 2: 225-234Google Scholar) andSyn-cytc6 and an ε556of 31.5 mm−1cm−1 for the ferrous form of cytf. For the assignment ofAna-cytc6, 0.7 mm15N-labeled and 1.0 mm unlabeled samples were prepared. To investigate complex formation betweenAna-cytc6 and cytf, microliter aliquots of a 1.9 mm cytf stock solution were titrated into an NMR sample containing 0.3 mm15N-Ana-cytc6. A reverse titration was also performed in which a sample containing 0.2 mm15N-Ana-cytc6 and 0.5 mm cytf was titrated with a 3.0 mmstock of unlabeled Ana-cytc6. To investigate complex formation betweenSyn-cytc6 and cytf, a 0.35 mm sample of 15N,13C-Syn-cytc6 was titrated with a 1.9 mm cytf solution. After each addition of protein, the pH of the samples was verified, and1H-15N HSQC spectra were recorded. For sequence-specific assignment of the backbone resonances of Ana-cytc6, two-dimensional 1H-15N HSQC, three-dimensional 1H-15N NOESY-HSQC (100 ms mixing time), and three-dimensional 1H-15N TOCSY-HSQC (80 ms mixing time) spectra were recorded on a Bruker DRX 500 NMR spectrometer. For assignment of the side-chain protons, two-dimensional homonuclear NOESY and TOCSY spectra were recorded. XWINNMR was used for spectral processing, and the assignment was performed in XEASY (23Bartels C. Xia T.-H. Billeter M. Güntert P. Wüthrich K. J. Biomol. NMR. 1995; 5: 1-10Google Scholar). Complete assignments of the 1H and15N resonances were determined for all backbone amides (see Supplementary material, Table S1). The non-exchangeable side-chain protons were also assigned. This assignment is consistent with those reported previously for Monoraphidium brauniicytc6 (24Banci L. Bertini I. De la Rosa M.A. Koulougliotis D. Navarro J.A. Walter O. Biochemistry. 1998; 37: 4831-4843Google Scholar) andSyn-cytc6 (25Beiβinger M. Sticht H. Sutter M. Ejchart A. Haehnel W. Rösch P. EMBO J. 1998; 17: 27-36Google Scholar). Measurements on samples containing cytf and either15N-Ana-cytc6 or15N,13C-Syn-cytc6 were performed on a Bruker DMX 600 NMR spectrometer operating at 300 K. Two-dimensional 1H-15N HSQC spectra (26Andersson P. Gsell B. Wipf B. Senn H. Otting G. J. Biomol. NMR. 1998; 11: 279-288Google Scholar) were recorded with spectral widths of 30.0 ppm (15N) and 13.9 ppm (1H). Analysis of the chemical-shift perturbation (ΔδBind) with respect to the free protein was performed in XEASY. Titration curves were obtained by plotting ΔδBind against the molar ratio (R) of [cytf]:[Ana-cytc6]. For the reverse titration, ΔδBind was plottedversus[Ana-cytc6]:[cytf]. Non-linear least-squares fits to a one-site model (27Kannt A. Young S. Bendall D.S. Biochim. Biophys. Acta-Bioenergetics. 1996; 1277: 115-126Google Scholar) were performed in Origin (Originlab, Northhampton, MA). This model explicitly treats the concentration of both proteins with R and ΔδBind as the independent and dependent variables, respectively (27Kannt A. Young S. Bendall D.S. Biochim. Biophys. Acta-Bioenergetics. 1996; 1277: 115-126Google Scholar). The binding constant (Ka) and the maximum chemical-shift change (ΔδMax) were the fitted parameters. A global fit was performed in which the curves were fitted simultaneously to a single Ka value, whereas ΔδMax was allowed to vary for each resonance. Comparison of1H-15N HSQC spectra of freeAna-cytc6 andAna-cytc6 in the presence of cytf revealed distinct differences arising from complex formation (Fig. 2). When cytfwas titrated into Ana-cytc6, 33 of the backbone amides experienced chemical-shift perturbation, ΔδBind ≥ 0.03 ppm (1H) and/or ≥ 0.10 ppm (15N). A single averaged resonance was observed for each backbone amide, indicating that the free and bound forms of cytc6 were in fast exchange on the NMR time scale. In addition to chemical-shift perturbation, the presence of cytf caused a general broadening of about 20 Hz of the amide resonances (Fig. 3A), as expected for complex formation (28Zuiderweg E.R.P. Biochemistry. 2002; 41: 1-7Google Scholar).Figure 3Line broadening effects. A, cross-sections along the F2 dimension through the 1HN resonance of Ser-16 inAna-cytc6. The resonance in the free protein (8.07 ppm) has a line width at half-height of 13 Hz. In the presence of two equivalents of cytf, the resonance shifts to 8.18 ppm, and the line width at half-height increases to 34 Hz.B, cross-sections along the F2dimension through the 1HN resonance of Gly-12 in Syn-cytc6. Despite the presence of two equivalents of cytf, the resonance is unperturbed (compare Ala-12 of Ana-cytc6, Fig. 4), and the line width at half-height increases by only 2 Hz.View Large Image Figure ViewerDownload (PPT) Titration curves of ΔδBind versus the molar ratio of cytf:Ana-cytc6were plotted for the 15N nuclei of the four most strongly shifted resonances (Fig. 4A). The curves clearly illustrate that the chemical-shift perturbation increases as a function of the cytf concentration. Despite the addition of two equivalents of cytf, however, saturation of the chemical-shift changes was not observed. The binding curves were fitted to a 1:1 model with a binding constant of ∼1 × 104m−1. The quality of the fit is poor, particularly at the beginning of the curve, which is perhaps due to a small systematic error in the determination of the protein ratio. To investigate this further, a reverse titration was performed in which a sample of cytf was titrated withAna-cytc6. In this case, the binding curves were fitted satisfactorily with a binding constant of 8 (±2) × 103m−1 (Fig. 4B). It can be concluded that the binding affinity of cytf for Ana-cytc6 is ∼104m−1, which is about 2 orders of magnitude greater than the affinity for the physiological partner, P. laminosum Pc (15Crowley P.B. Otting G. Schlarb-Ridley B.G. Canters G.W. Ubbink M. J. Am. Chem. Soc. 2001; 123: 10444-10453Google Scholar, 29Crowley P.B. Rabe K.S. Worrall J.A.R. Canters G.W. Ubbink M. ChemBioChem. 2002; 3: 526-533Google Scholar). From the ratio of the observed ΔδBind to the fitted ΔδMax(Fig. 4B), it was calculated that 63% of theAna-cytc6 was bound in the first point of the reverse titration. Six structures of cytc6, from green algal (30Kerfeld C.A. Anwar H.P. Interrante R. Merchant S. Yeates T.O. J. Mol. Biol. 1995; 250: 627-647Google Scholar, 31Schnackenberg J. Than M.E. Mann K. Wiegand G. Huber R. Reuter W. J. Mol. Biol. 1999; 290: 1019-1030Google Scholar, 32Yamada S. Park S.-Y. Shimizu H. Koshizuka Y. Kadokura K. Satoh T. Suruga K. Ogawa M. Isogai Y. Nishio T. Shiro Y. Oku T. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 1577-1582Google Scholar), red algal (33Sawaya M.R. Krogmann D.W. Serag A. Ho K.K. Yeates T.O. Kerfeld C.A. Biochemistry. 2001; 40: 9215-9225Google Scholar), and cyanobacterial (12Frazão C. Soares C.M. Carrondo M.A. Pohl E. Dauter Z. Wilson K.S. Hervas M. Navarro J.A. De la Rosa M.A. Sheldrick G.M. Structure. 1995; 3: 1159-1169Google Scholar, 25Beiβinger M. Sticht H. Sutter M. Ejchart A. Haehnel W. Rösch P. EMBO J. 1998; 17: 27-36Google Scholar) sources, have been determined previously. All of the known structures exhibit high structural homology. At present, the structure ofAna-cytc6 is unavailable, and therefore, a model was built in Swiss-MODEL (34Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Google Scholar) using the NMR structure of Syn-cytc6 (25Beiβinger M. Sticht H. Sutter M. Ejchart A. Haehnel W. Rösch P. EMBO J. 1998; 17: 27-36Google Scholar) as a template. The chemical-shift map in Fig. 5 illustrates the location of the affected residues in this model with each residue coloredaccording to its observed ΔδBind. The complex interface consists of a well defined patch, which surrounds the exposed methyl groups (methyl 3, thioether 4 methyl, and methyl 5) of the heme. This patch is composed mainly of three stretches of the primary structure, residues 9–19, 23–26, and 51–61. The first of these includes the heme-binding motif CXYCH, whereas the sixth ligand, Met-58, occurs in the third stretch. Val-25, which was shown to be important for the interaction between cytc6 and PSI (8Molina-Heredia F.P. Dı́az-Quintana A. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 1999; 274: 33565-33570Google Scholar), is found in the second stretch. Cys-17, Met-58, and Ala-60 experience the largest shifts in the complex. Alanine and asparagine are the most abundant residues in the interface, accounting for one-third of all the affected residues. Although 60% of the interface can be classified as hydrophobic, only 4 residues are charged (Lys-55, Lys-66, Glu-68, and Lys-80). Notably the conserved arginine, Arg-64, which has been implicated in the reaction with PSI (9Molina-Heredia F.P. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 2001; 276: 601-605Google Scholar), was not shifted in the complex. Fragata (35Fragata M. Biophys. J. 2002; 82: 1618Google Scholar) has produced a theoretical model of the complex formed between M. brauniicytc6 and turnip cytf (Protein Data Bank accession code 1jx8). There is good agreement between the binding site on cytc6 identified in this model and the experimentally observed interaction site onAna-cytc6. The complex of cytf andAna-cytc6 was also investigated at 50, 100, and 200 mm NaCl. The observed ΔδBind for the 33 affected amides is plotted as a function of the salt concentration in Fig. 6. As the salt concentration was increased, ΔδBind decreased. At 200 mmNaCl, the ΔδBind is zero for most residues. This suggests that the binding constant is considerably reduced, illustrating the importance of attractive electrostatic interactions in complex formation. The more strongly affected residues such as Cys-17 and Met-58 still experience appreciable shifts at 200 mmNaCl. Furthermore, although the line broadening decreased with increasing salt, there remains ∼5 Hz broadening at 200 mm NaCl. These observations are indicative of a significant interaction even at high ionic strength. In contrast to the case of Ana-cytc6, titration of cytf intoSyn-cytc6 produced only minor effects in the 1H-15N HSQC spectra. Despite the presence of two equivalents of cytf, the amide resonances ofSyn-cytc6 did not experience chemical-shift perturbation. Line broadening effects on the order of 2 Hz were observed (Fig. 3B), consistent with a weak and highly dynamic interaction (36Worrall J.A.R. Liu Y. Crowley P.B. Nocek J.M. Hoffman B.M. Ubbink M. Biochemistry. 2002; 41: 11721-11730Google Scholar). Increasing the ionic strength by the addition of 200 mm NaCl had no effect on the protein interactions. In contrast to the clearly defined complex between cytf and Ana-cytc6, there is no evidence for specific complex formation withSyn-cytc6. From our results, it is clear thatAna-cytc6 and cytf form a well defined complex and that the amount of complex formed is dependent on the ionic strength. It has been shown previously thatAna-cytc6 forms a complex withAnabaena PSI, the affinity of which decreases with increasing ionic strength (7Hervás M. Navarro J.A. Dı́az A. De la Rosa M.A. Biochemistry. 1996; 35: 2693-2698Google Scholar). AlthoughAna-cytc6 has a net charge close to zero, the presence of positive residues in the vicinity of the heme group promotes favorable electrostatic docking to cytf. The role of electrostatics in this complex is therefore analogous to the complex formed between plant cytf and Pc in vitro(14Ubbink M. Ejdeback M. Karlsson B.G. Bendall D.S. Structure. 1998; 6: 323-335Google Scholar, 27Kannt A. Young S. Bendall D.S. Biochim. Biophys. Acta-Bioenergetics. 1996; 1277: 115-126Google Scholar, 37Soriano G.M. Ponamarev M.V. Piskorowski R.A. Cramer W.A. Biochemistry. 1998; 37: 15120-15128Google Scholar, 38Bergkvist A. Ejdeback M. Ubbink M. Karlsson G. Protein Sci. 2001; 10: 2623-2626Google Scholar, 39Soriano G.M. Ponamarev M.V. Tae G.-S. Cramer W.A. Biochemistry. 1996; 35: 14590-14598Google Scholar). As illustrated in Fig. 5, the interaction site ofAna-cytc6 is composed mainly of hydrophobic residues, which is necessary to achieve specific complex formation between the two proteins. Such a hydrophobic site is similar to that proposed previously for the interaction ofAna-cytc6 with PSI (8Molina-Heredia F.P. Dı́az-Quintana A. Hervás M. Navarro J.A. De la Rosa M.A. J. Biol. Chem. 1999; 274: 33565-33570Google Scholar). A type I mechanism has been reported for the reduction of PSI bySyn-cytc6 (21Sutter M. Sticht H. Schmid R. Hörth P. Rösch P. Haehnel W. Mathis P. Photosynthesis: From Light to Biosphere. II. Kluwer Academic Publishers, The Netherlands1995: 563-566Google Scholar). In this mechanism, the electron transfer rate is proportional to the number of collisions in which the redox centers of both proteins are aligned. The NMR titration of cytf intoSyn-cytc6 provides no evidence for specific complex formation but suggests a highly dynamic interaction between these partners, in agreement with a type I mechanism. The optimal orientation for productive collisions is facilitated by the prominent acidic patch on the backside ofSyn-cytc6 (Fig. 1) since the front face will be the energetically more favorable approach with the acidic partners, cytf and PSI. Apparently, cytc6 from different organisms utilizes different mechanisms for the electron transfer reaction with its partners. A similar conclusion has been reached for the interactions of cytf and Pc from different organisms (15Crowley P.B. Otting G. Schlarb-Ridley B.G. Canters G.W. Ubbink M. J. Am. Chem. Soc. 2001; 123: 10444-10453Google Scholar). The source of this different reactivity can be traced to the nature of the protein surfaces and ultimately to variations in the primary structures. The sequences of Ana-cytc6 andSyn-cytc6 share 67% identity. Of the 33 residues identified in theAna-cytc6 interaction site, 22 are conserved in the Syn-cytc6 sequence. Only 6 residues, located around the periphery of the complex interface, are significantly different inSyn-cytc6. Ala-19 and Gln-26 are replaced by methionines, which have been implicated as endogenous antioxidants (25Beiβinger M. Sticht H. Sutter M. Ejchart A. Haehnel W. Rösch P. EMBO J. 1998; 17: 27-36Google Scholar, 40Levine R.L. Mosoni L. Berlett B.S. Stadtman E.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15036-15040Google Scholar). Ala-49 is replaced by a tyrosine, whereas glutamine and histidine replace Thr-52 and Asn-53, respectively. This cluster of three variations inSyn-cytc6 results in a bulkier surface, which may hinder interactions with cytf. The replacement of Lys-66 by a threonine inSyn-cytc6 is representative of the most striking difference between the two sequences. Five additional lysines in Ana-cytc6 contribute to the significantly higher pI of this protein and enable attractive electrostatic interactions with the acidic cytf. The different roles of electrostatics and hydrophobics can be further illustrated by comparison with the recently characterized, non-physiological complex of yeast cytochrome c(cytc) and cytf (29Crowley P.B. Rabe K.S. Worrall J.A.R. Canters G.W. Ubbink M. ChemBioChem. 2002; 3: 526-533Google Scholar). It was found that cytf binds two equivalents of cytc with binding constants of ∼2 × 104m−1and 4 × 103m−1. The higher binding affinity, as compared with the cytf-Ana-cytc6 complex, probably arises from the strong electrostatic attraction between cytf and cytc, which has a pI of 9.7 (41Park K.S. Frost B.F. Shin S. Park I.K. Kim S. Paik W.K. Arch. Biochem. Biophys. 1988; 267: 195-204Google Scholar). Fewer residues experienced chemical-shift perturbation in this complex, resulting in a less extensive interaction site (29Crowley P.B. Rabe K.S. Worrall J.A.R. Canters G.W. Ubbink M. ChemBioChem. 2002; 3: 526-533Google Scholar) (Fig. 7, A and B). Furthermore, the chemical-shift perturbation was on average 50% smaller than that observed in the complex withAna-cytc6. The prevalence of electrostatic attractions may reduce the amount of time spent in a single orientation, as determined by hydrophobic contacts, and consequently, fewer and smaller chemical-shift changes are observed. Alternatively, the diminished interaction site observed for cytc may be explained in terms of “goodness of fit.” It can be assumed that the hydrophobic patch surrounding the heme is better adapted in Ana-cytc6 than in yeast cytc for binding to cytf. This complimentarity favors closer contact between the protein surfaces and thus a larger interaction site. Protein docking simulations, using an NMR filter implemented in BiGGER (42Palma P.N. Krippahl L. Wampler J.E. Moura J.J.G. Proteins. 2000; 39: 372-384Google Scholar), identified the cytc binding sites as the front (Site I) and back (Site II) faces of the heme region of cytf(29Crowley P.B. Rabe K.S. Worrall J.A.R. Canters G.W. Ubbink M. ChemBioChem. 2002; 3: 526-533Google Scholar) (Fig. 7C). A similar docking simulation using theAna-cytc6 model gave slightly different results. Although the front face of the heme remains the favored site of interaction, there is a significant fraction of favorable docking orientations beneath the heme region in the large domain of cytf (Fig. 7D). Notably, there is significant overlap between the top-ranking docking configuration found for Ana-cytc6 in BiGGER and the docking orientation of M. brauniicytc6 in the model of Fragata (35Fragata M. Biophys. J. 2002; 82: 1618Google Scholar). In this model, between the algal cytc6 and plant cytf, there are favorable electrostatics between complimentary charged patches, a ridge of lysines on the small domain of cytf and a cluster of acidic residues on the side of cytc6. This results in a slightly different orientation of cytc6 in comparison with the cyanobacterial complex, which does not possess such well defined complimentary charged patches. P. B. Crowley is grateful to Dr. J. A. R. Worrall for helpful discussions and to C. Erkelens for assistance with the NMR facilities. A. Dı́az-Quintana and P. Nieto thank Dr. M. Bruix for help with recording NMR spectra for the assignment of Ana-cytc6. Dr. M. Hervás is kindly acknowledged for helpful discussions and for critical reading of the manuscript. Download .pdf (.07 MB) Help with pdf files
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