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Cytochrome f is a unique, integral membrane protein. The background to its discovery by Robert Hill (1899-1991) and Ronald Scarisbrick over 60 years ago and the influence of David Keilin (1887-1963) and Frederick Gowland Hopkins (1861-1947) are discussed. The development of methods for isolating cytochrome f is outlined, emphasizing the remarkable achievement of Hill and Scarisbrick at a time when few if any membrane proteins had been isolated, and the importance of the discovery of a natural proteolysis in Brassica spp., stimulated by organic solvents, by Eijiro Yakushiji and coworkers and by Masa-aki Takahashi and Kozi Asada in 1975. The significance of different types of instrumentation in the study of cytochrome f is discussed, drawing attention to the importance of the microspectroscope ocular for its discovery, to types of spectrophotometer developed especially by Britton Chance for spectrophotometric measurements on turbid suspensions of cells and plastids, and to the history of stopped-flow spectrophotometry. The stopped-flow instrument originated in the bucket-scale flow methods of Hartridge and Roughton (1923), and was later developed on the microscale by Chance. Finally, the problems that remain for understanding the behavior of cytochrome f in the thylakoid lumen are contrasted with the significance of in vitro studies that provide a paradigm for transient protein-protein interactions in the wider field of biology as a whole.
Direct cyclic voltage-current responses, produced in the absence of redox mediators, for two detergent-solubilized integral membrane proteins, spinach cytochrome f and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.
The complex of yeast cytochrome c and cytochrome f from the cyanobacterium Phormidium laminosum was investigated by NMR spectroscopy. Chemical shift perturbation analysis reveals that residues around the haem edge of cytochrome c are involved in the complex interface. Binding curves derived from an NMR spectroscopy titration at 10 mM ionic strength indicate that there are two sites for cytochrome c with binding constants of approximately 2 x 10(4) M(-1) and 4 x 10(3) M(-1). A protein docking simulation with NMR-derived constraints identifies two sites, at the front (Site I) and back faces (Site II) of the haem region of cytochrome f. Site I is homologous to the binding site previously determined for the natural cytochrome f partner plastocyanin. Site II may represent the binding site for the Rieske protein in the cytochrome bf complex. Cytochrome c and plastocyanin are shown to compete for binding at Site I. The competition appears to involve electrostatic screening rather than simple steric occlusion of the binding site.
The physiological transient complex between cytochrome f (Cf) and cytochrome c(6) (Cc(6)) from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by NMR spectroscopy. The binding constant at low ionic strength is 8 +/- 2 mM(-1), and the binding site of Cc(6) for Cf is localized around its exposed haem edge. On the basis of the experimental data, the resulting docking simulations suggest that Cc(6) binds to Cf in a fashion that is analogous to that of plastocyanin but differs between prokaryotes and eukaryotes.
Noninvasive reconstitution of the heme in cytochrome c(6) with zinc(II) ions allowed us to study the photoinduced electron-transfer reaction (3)Zncyt c(6) + cyt f(III) --> Zncyt c(6)(+) + cyt f(II) between physiological partners cytochrome c(6) and cytochrome f, both from Chlamydomonas reinhardtii. The reaction kinetics was analyzed in terms of protein docking and electron transfer. In contrast to various protein pairs studied before, both the unimolecular and the bimolecular reactions of this oxidative quenching take place at all ionic strengths from 2.5 through 700 mM. The respective intracomplex rate constants are k(uni) (1.2 +/- 0.1) x 10(4) s(-1) for persistent and k(bi) (9 +/- 4) x 10(2) s(-1) for the transient protein complex. The former reaction seems to be true electron transfer, and the latter seems to be electron transfer gated by a structural rearrangement. Remarkably, these reactions occur simultaneously, and both rate constants are invariant with ionic strength. The association constant K(a) for zinc cytochrome c(6) and cytochrome f(III) remains (5 +/- 3) x 10(5) M(-1) in the ionic strength range from 700 to 10 mM and then rises slightly to (7 +/- 2) x 10(6) M(-1), as ionic strength is lowered to 2.5 mM. Evidently, docking of these proteins from C. reinhardtii is due to hydrophobic interaction, slightly augmented by weak electrostatic attraction. Kinetics, chromatography, and cross-linking consistently show that cytochrome f self-dimerizes at ionic strengths of 200 mM and higher. Cytochrome f(III) quenches triplet state (3)Zncyt c(6), but its dimer does not. Formation of this unreactive dimer is an important step in the mechanism of electron transfer. Not only association between the reacting proteins, but also their self-association, should be considered when analyzing reaction mechanisms.
Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of (35)S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system.
Cytochrome f and plastocyanin are redox partners in the photosynthetic electron-transfer chain. Electron transfer from cytochrome f to plastocyanin occurs in a specific short-lived complex. To obtain detailed information about the binding interface in this transient complex, the effects of binding on the backbone and side-chain protons of plastocyanin have been analyzed by mapping NMR chemical-shift changes. Cytochrome f was added to plastocyanin up to 0.3 M equiv, and the plastocyanin proton chemical shifts were measured. Out of approximately 500 proton resonances, 86% could be observed with this method. Nineteen percent demonstrate significant chemical-shift changes and these protons are located in the hydrophobic patch (including the copper ligands) and the acidic patches of plastocyanin, demonstrating that both areas are part of the interface in the complex. This is consistent with the recently determined structure of the complex [Ubbink, M., Ejdebäck, M., Karlsson, B. G., and Bendall, D. S. (1998) Structure 6, 323-335]. The largest chemical-shift changes are found around His87 in the hydrophobic patch, which indicates tight contacts and possibly water exclusion from this part of the protein interface. These results support the idea that electron transfer occurs via His87 to the copper in plastocyanin and suggest that the hydrophobic patch determines the specificity of the binding. The chemical-shift changes in the acidic patches are significant but small, suggesting that the acidic groups are involved in electrostatic interactions but remain solvent exposed. The existence of small differences between the present data and those used for the structure may imply that the redox state of the metals in both proteins slightly affects the structure of the complex. The chemical-shift mapping is performed on unlabeled proteins, making it an efficient way to analyze effects of mutations on the structure of the complex.
BACKGROUND: Cytochrome f is the high potential electron acceptor of the chloroplast cytochrome b6f complex, and is the electron donor to plastocyanin. The 285-residue cytochrome f subunit is anchored in the thylakoid membrane of the chloroplast by a single membrane-spanning segment near the carboxyl terminus. A soluble redox-active 252-residue lumen-side polypeptide with native spectroscopic and redox properties, missing the membrane anchor and carboxyl terminus, was purified from turnip chloroplasts for structural studies. RESULTS: The crystal structure of cytochrome f, determined to 2.3 A resolution, has several unexpected features. The 252-residue polypeptide is organized into one large and one small domain. The larger heme-binding domain is strikingly different from known structures of other c-type cytochromes and has the same fold as the type III domain of the animal protein, fibronectin. Cytochrome f binds heme with an unprecedented axial heme iron ligand: the amino terminus of the polypeptide. CONCLUSION: The first atomic structure of a subunit of either the cytochrome b6f complex or of the related cytochrome bc1 complex has been obtained. The structure of cytochrome f allows prediction of the approximate docking site of plastocyanin and should allow systematic studies of the mechanism of intra- and inter-protein electron transfer between the cytochrome heme and plastocyanin copper, which are approximately isopotential. The unprecedented axial heme iron ligand also provides information on the sequence of events (i.e. cleavage of signal peptide and ligation of heme) associated with translocation of the cytochrome across the membrane and its subsequent folding.
Using Brownian dynamics simulations, all of the charged residues in Chlamydomonas reinhardtii cytochrome c(6) (cyt c(6)) and plastocyanin (PC) were mutated to alanine and their interactions with cytochrome f (cyt f) were modeled. Systematic mutation of charged residues on both PC and cyt c(6) confirmed that electrostatic interactions (at least in vitro) play an important role in bringing these proteins sufficiently close to cyt f to allow hydrophobic and van der Waals interactions to form the final electron transfer-active complex. The charged residue mutants on PC and cyt c(6) displayed similar inhibition classes. Our results indicate a difference between the two acidic clusters on PC. Mutations D44A and E43A of the lower cluster showed greater inhibition than do any of the mutations of the upper cluster residues. Replacement of acidic residues on cyt c(6) that correspond to the PC's lower cluster, particularly E70 and E69, was observed to be more inhibitory than those corresponding to the upper cluster. In PC residues D42, E43, D44, D53, D59, D61, and E85, and in cyt c(6) residues D2, E54, K57, D65, R66, E70, E71, and the heme had significant electrostatic contacts with cyt f charged residues. PC and cyt c(6) showed different binding sites and orientations on cyt f. As there are no experimental cyt c(6) mutation data available for algae, our results could serve as a good guide for future experimental work on this protein. The comparison between computational values and the available experimental data (for PC-cyt f interactions) showed overall good agreement, which supports the predictive power of Brownian dynamics simulations in mutagenesis studies.
The cytochrome b(6)f complex is an obligatory electron transfer and proton-translocating enzyme in all oxygenic photosynthesis. Its operation has been described by the "Q-cycle." This model proposes that electrons are transferred from plastoquinol to plastocyanin (the reductant of P700 in Photosystem I) through, obligatorily in series, the iron-sulfur and the cytochrome f redox centers in the cytochrome b(6)f complex. However, here we demonstrate that (a) the iron-sulfur center-dependent reductions of plastocyanin and P700 are much faster than cytochrome f reduction, both in Chlamydomonas reinhardtii cytochrome f mutants and in the wild type, and (b) the steady-state photosynthetic electron transport does not correlate with strongly inhibited cytochrome f reduction kinetics in the mutants. Thus, cytochrome f is not an obligatory intermediate for electrons flowing through the cytochrome b(6)f complex. The oxidation equivalents from Photosystem I are delivered to the high potential chain of the cytochrome b(6)f complex both at the cytochrome f level and, independently, at another site connected to the quinol-oxidizing site, possibly the iron-sulfur center.
The 1.96 A structure of turnip cytochrome f revealed a linear internal chain of H2O molecules with the oxygen atoms of the chain having occupancies and "B" factors comparable to those of neighboring atoms [Martinez et al. (1996) Protein Sci. 5, 1081-1092. ]. Four waters extend 11 A from the heme toward Lys66 on the cytochrome surface. All residues that contribute an atom to the 15 H-bonds of five internal H2O molecules are essentially conserved in 23 cytochrome sequences. With only Gln and Asn side chains involved in H-bonding, the water chain resembles a "proton wire". The function of the conserved H2O chain was tested through site-directed mutagenesis of these Asn and Gln residues. Four of the five conserved Asn/Gln residues were changed in six mutants generated in the green alga, Chlamydomonas reinhardtii. Except for the N168F mutant, all grew photosynthetically. Although the rates of oxidation of cyt f oxidation and of reduction of cyt b6 (5-6 ms in the wild type) were not significantly affected, the rates of cyt f reduction and generation of the slow electrochromic band shift (Deltapsis) were markedly decreased, the half-times increasing to as much as 38 and 18 ms, respectively. Thus, in these mutants, reduction of cyt b6 reduction clearly precedes that of cyt f. Retardation of Deltapsis in the absence of an observable change in the rate of cyt b6 reduction implied that the rate of H+ translocation decreased in the mutants, and electron transfer was concomitantly retarded, most likely between the ISP and cyt f. The following was concluded: (i) proton and electron transfer are coupled in reduction of cyt f, and the cyt f water chain functions in H+ transfer; (ii) reduction of the high- and low-potential chains in the b6f complex is not concerted in the water chain mutants; and (iii) quinol deprotonation and electron transfer from reduced quinone are initiated by an early event, probably the movement of the ISP triggered by oxidation of cyt f.
The interaction of Chlamydomonas cytochrome f (cyt f) with either Chlamydomonas plastocyanin (PC) or Chlamydomonas cytochrome c(6) (cyt c(6)) was studied using Brownian dynamics simulations. The two electron acceptors (PC and cyt c(6)) were found to be essentially interchangeable despite a lack of sequence homology and different secondary structures (beta-sheet for PC and alpha-helix for cyt c(6)). Simulations using PC and cyt c(6) interacting with cyt f showed approximately equal numbers of successful complexes and calculated rates of electron transfer. Cyt f-PC and cyt f-cyt c(6) showed the same types of interactions. Hydrophobic residues surrounding the Y1 ligand to the heme on cyt f interacted with hydrophobic residues on PC (surrounding the H87 ligand to the Cu) or cyt c(6) (surrounding the heme). Both types of complexes were stabilized by electrostatic interactions between K65, K188, and K189 on cyt f and conserved anionic residues on PC (E43, D44, D53, and E85) or cyt c(6) (E2, E70, and E71). Mutations on cyt f had identical effects on its interaction with either PC or cyt c(6). K65A, K188A, and K189A showed the largest effects whereas residues such as K217A, R88A, and K110A, which are located far from the positive patch on cyt f, showed very little inhibition. The effect of mutations observed in Brownian dynamics simulations paralleled those observed in experiments.
The results of a comprehensive Q-band resonance Raman investigation of cytochrome c1 and cytochrome f subunits of bc1 and b6f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.
In oxygenic photosynthesis the cytochrome bf complex links electron transport between photosystem II and photosystem I. The largest subunit of the complex is cytochrome f, a 32-kDa polypeptide that is anchored in the membrane by a transmembrane alpha helix located near the carboxyl end. The three-dimensional structure of the soluble domain of cytochrome f isolated from turnip has been determined by X-ray crystallography to 1.96 A resolution. The structure revealed several novel features compared to previously solved soluble c-type cytochrome structures, including a predominant beta-strand motif, the N-terminal alpha-amino group of a tyrosyl residue as an orthogonal ligand, and a bound internal water chain. Here we report a novel and unprecedented extinction coefficient for cytochrome f. Using the pyridine hemochrome assay, the reduced minus oxidized extinction coefficient for the soluble domain of turnip cytochrome f was 26 +/- 1 mM-1 cm-1 for the alpha-band wavelength peak at 554 nm relative to the isosbestic wavelengths (534, 543.5 and 560.5 nm), and 25 +/- 1 mM-1 cm-1 for spinach cytochrome f relative to the isosbestic wavelengths (533.5, 543.3 and 560.2). The extinction coefficients reported here are significantly higher than previously published values for cytochrome f. We believe earlier determinations underestimated the cytochrome f extinction coefficient and that the same is likely true for commonly used extinction coefficients of cytochrome b6. The cytochrome f extinction coefficient is large compared to most other c-type cytochromes, which could be due to the unique axial ligand of the cytochrome f heme. Polarographic measurements show the midpoint potential of soluble turnip cytochrome f to be 362 +/- 5 mV at pH 7.5. The midpoint potential was pH-independent from 5.0 to 8.5, and pH-dependent from pH 8.5 to 10.5 (-58 mV/pH unit) with a pK on the oxidized from near 9. Storage of some samples of purified turnip and spinach cytochrome f at -20 degrees C modified the heme environment in a fraction of the protein, shifting the midpoint potential to near -165 mV (pH 7.5) and the peak of the alpha-band absorption spectrum from 554 nm to 552 nm.
Cytochrome f of oxygenic photosynthesis has an unprecedented structure, including the N-terminus being a heme ligand. The adjacent N-terminal heme-shielding domain is enriched in aromatic amino acids. The atomic structures of the chloroplast and cyanobacterial cytochromes f were compared to explain spectral and redox differences between them. The conserved aromatic side chain in the N-terminal heme-shielding peptide at position 4, Phe and Tyr in plants and algae, respectively, and Trp in cyanobacteria, is in contact with the heme. Mutagenesis of cytochrome f from the eukaryotic green alga Chlamydomonas reinhardtii showed that a Phe4 --> Trp substitution in the N-terminal domain was unique in causing a red shift of 1 and 2 nm in the cytochrome Soret (gamma) and Q (alpha) visible absorption bands, respectively. The resulting alpha band peak at 556 nm is characteristic of the cyanobacterial cytochrome. Conversely, a Trp4 --> Phe mutation in the expressed cytochrome from the cyanobacterium Phormidium laminosum caused a blue shift to the 554 nm alpha band peak diagnostic of the chloroplast cytochrome. Residue 4 was found to be the sole determinant of this 60 cm(-)(1) spectral shift, and of approximately one-half of the 70 mV redox potential difference between cytochrome f of P. laminosum and C. reinhardtii (E(m7) = 297 and 370 mV, respectively). The proximity of Trp-4 to the heme implies that the spectral and redox potential shifts arise through differential interaction of its sigma- or pi-electrostatic potential with the heme ring and of the pi-potential with the heme Fe orbitals, respectively. The dependence of the visible spectrum and redox potential of cytochrome f on the identity of aromatic residue 4 provides an example of the use of the relatively sharp cytochrome spectrum as a "spectral fingerprint", and of the novel structural connection between the heme and a single nonliganding residue.
The photo-oxidation of cytochrome f (cytochrome c(554)) in bundle sheath cells isolated from leaves of maize (Zea mays var. DS 606A) has been compared with that in intact maize leaf and in isolated pea leaf cells (Pisum sativum L.). In all cases, illumination with red light caused a negative absorbance change at 554 nm which was attributed to the oxidation of cytochrome f. The extent of this change was greater using monochromatic red light at wavelengths above 700 nm compared with wavelengths below 700 nm. 3-(3,4-Dichlorophenyl)-1, 1-dimethylurea abolished this difference in bundle sheath cells. After illumination for 1 minute or longer in bundle sheath cells, reduction of cytochrome f in the dark was rapid only if the wavelength of the illuminating light was below 700 nm. In the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethlyurea, reduction was slow after illumination at all wavelengths.Cytochrome f photo-oxidation was also followed in cells of a mutant of Chlamydomonas reinhardi, ac-21, which has isolated chloroplasts that exhibit photochemical reactions similar to those shown by isolated bundle sheath chloroplasts. No evidence was obtained for photoreduction of cytochrome f in the mutant.It was concluded that in the chloroplast of the intact bundle sheath cell of maize there is electron flow between photosystem II and cytochrome f resulting in photoreduction of the cytochrome.
The interaction between cytochrome f and its electron acceptor plastocyanin (PC) was studied. To address the question of which specific regions and which of the positively charged residues of cytochrome f are important for the interaction with the negatively charged residues of PC we have used two different experimental approaches. Cytochrome f was proteolytically cleaved and fragments that could bind to a PC-affinity column were isolated. The smallest of these fragments was analysed to give information on the minimum structural requirement for binding to PC. By this procedure, we identified a peptide of approx. 11 kDa, containing the heme binding site, and having an N-terminal sequence identical to that of the mature cytochrome f. This finding suggests that the first 90 amino acids of cytochrome f contain at least some of the residues interacting with PC. The second approach involved modification of Arg residues of cytochrome f with the specific chemical modifier, hydroxyphenylglyoxal (HPG). Cytochrome f modification was performed in the absence of PC to enable identification of residues that are protected from modification when PC is bound to cytochrome f. Two peptides containing Arg residues which are modified in the absence of PC, but are not modified when PC is present, were isolated. Sequence analysis of these two peptides revealed that Arg residues no. 88 and 154 of cytochrome f are the residues that are protected from modification when cytochrome f is bound to PC, suggesting a role for these residues in the binding of cytochrome f to PC.