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Volume 271, Number 11, Issue of March 15, 1996 pp. 6225-6232
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
N-terminal Mutants of Chloroplast Cytochrome f
EFFECT ON REDOX REACTIONS AND GROWTH IN CHLAMYDOMONAS REINHARDTII(*)

(Received for publication, November 17, 1995; and in revised form, January 9, 1996)

Jianhui Zhou ,  Javier G. Fernández-Velasco ,  Richard Malkin (§)

From the Department of Plant Biology, University of California, Berkeley, California 94720-3102

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The N-terminal tyrosine of cytochrome f, which provides the sixth ligand to the heme group, has been changed by site-directed mutagenesis in Chlamydomonas reinhardtii to evaluate the role of this amino acid in assembly and function. The second and third residues, proline and valine, respectively, have also been mutated. Y1P is the only strain that did not grow photoautotrophically. The other strains show cytochrome b(6)f complex/photosystem I reaction center chlorophyll, photosystem I unit size and chlorophyll a+b/cell ratios comparable with wild-type cells. Rates of cytochrome f photooxidation in all strains were similar (t 300 µs), whereas the rate of re-reduction sensitive to stigmatellin (at E(h) = 0 mV, (where E(h) is the ambient redox potential) for wild-type, Y1W, Y1F, Y1S, P2V, and V3P had a t of 3, 4, 5, 9, 40, and 2 ms, respectively. Rates of oxygen evolution by whole cells of P2V, Y1F, and Y1S were 67, 80, and 80% of wild-type rates, respectively. At low light intensity, all competent strains had the same growth rate whereas at saturating intensities, only P2V showed a significant inhibition. These results are considered in relation to structure-function relationships in the cytochrome f molecule.

INTRODUCTION

The cytochrome b(6)f complex functions as a plastoquinol-plastocyanin oxidoreductase in the membranes of all oxygenic photosynthetic organisms(1, 2, 3) . The reactions of this integral membrane protein complex are analogous to those of the mitochondrial cytochrome bc(1) complex, and this analogy extends to their composition(1, 4) . Among the electron transfer carriers in the cytochrome b(6)f complex is a high-potential c-type cytochrome, known as cytochrome f, that serves as the electron donor to plastocyanin. Analysis of the cytochrome f protein has shown that it contains approximately 285 amino acid residues and has a molecular mass of approximately 31 kDa(5, 6) . Hydropathy profiles, based on derived DNA sequences, has predicted one transmembrane spanning region, near the C terminus of the protein, and that the bulk of the protein, some 250 amino acids, is located in the interior space, the lumen, of the thylakoid membrane(6) . The single heme group is bound near the N terminus of the protein. Support for this model was originally obtained by Wiley et al.(7) in studies of the proteolysis of the protein using intact thylakoids.

Recently, Martinez et al. (8) have reported a high resolution (2.3 Å) three-dimensional structure of the lumenal, water-soluble portion of cytochrome f from turnip, and this structure has revealed some unusual features. The overall structure shows that cytochrome f contains two major domains, a large lower region that shows extensive beta-sheet structure and a smaller upper region that contains several surface-exposed basic amino acids presumed to function in the interaction of cytochrome f with its acidic electron transfer partner, plastocyanin. One of the most unusual findings, however, came when the ligation of the heme group was elucidated. It was known that the cytochrome f heme was covalently bound to the protein by thioether bonds through two highly conserved cysteine residues (Cys and Cys) and that the fifth ligand to the heme was a proximal histidine group (His), but the nature of the sixth ligand was unknown. Early spectroscopic measurements had indicated that a nitrogen group served as the sixth ligand and led to the proposal that Lys was the axial ligand(9) . The x-ray structure showed that the sixth ligand to the heme was actually the alpha-amino group of Tyr^1, the N terminus of the protein. Other c-type cytochromes are known that contain either histidine or methionine groups as the axial heme ligand. The heme group of cytochrome f is located in a hydrophobic pocket that is shielded from the solvent. The pathway of electron transfer into and out of the heme is of great interest. The position of Tyr^1 in the structure shows that the aromatic ring lies parallel to the plane of the heme ring, suggesting a possible role in electron transfer through the aromatic ring to the iron of the heme group. Other amino acid side chains involved in shielding the heme from the solvent are Pro^2, Phe^4, Pro, and Pro, all of which form the hydrophobic pocket.

We have been studying structure-function relationships in the cytochrome b(6)f complex and have recently initiated site-directed mutagenesis studies of the individual subunits of the complex using the green alga, Chlamydomonas reinhardtii. We have reported previously the cloning and sequencing of the cytochrome f gene, petA, from C. reinhardtii(10) , and in this work, we have used this gene in conjunction with the chloroplast transformation system described for this organism (11, 12) to prepare site-directed mutants of cytochrome f. We have examined effects of mutations in the N-terminal region of cytochrome f on the time-resolved kinetics of oxidation and reduction of the protein. Because of the unique properties of the N-terminal tyrosine residue, a number of mutants have been prepared in which this residue has been replaced by other amino acids. In addition, changes in Pro^2 and Val^3 have been examined. We find that mutants in the Tyr^1 position show comparatively small effects in terms of electron transport and growth, while a Pro^2 mutant is markedly affected. These results are discussed in terms of structural and functional aspects of the cytochrome f molecule.

MATERIALS AND METHODS

Strains and Culture Conditions

C. reinhardtii wild-type strain (7d) was obtained from Dr. Michel Goldschmidt-Clermont (University of Geneva, Switzerland). Wild-type and all phototrophic strains were grown in HS medium (13) at 20 °C under high light (350 µE m s) and acetate-requiring mutants were grown in HSHA medium (HS medium supplemented with 4 g/liter of sodium acetate) under dim light (5 µE m s). For liquid cultures grown under phototrophic condition, carbon dioxide was provided by bubbling the culture with a mixture of 3% CO(2), 97% air. Cells used for both DNA isolation and transformation were harvested during the mid-exponential phase of growth. Growth rates were monitored by measuring the absorbance at 750 nm, a wavelength at which chlorophyll interference is minimal(14) . For growth on solid media, HS or HSHA media was prepared with 1.5% Bacto-agar (Difco). Selection for antibiotic resistance was accomplished by supplementing the medium with spectinomycin (150 µg/ml) as required. For cells to be used for chloroplast transformation, 0.5 mM 5-fluorodeoxyuridine was included in the growth medium to reduce the copy number of the chloroplast genomes (15) .

DNA Manipulations

Chloroplast DNA was isolated from C. reinhardtii as described previously(16) . Plasmid DNAs were isolated from Escherichia coli strains as described by Sambrook et al.(17) . Restriction enzymes and other DNA modifying enzymes were obtained from Life Technologies, Inc. or New England Biolabs (Beverley, MA) and used according to the recommendation of the manufacturers. Taq DNA polymerase used for polymerase chain reaction reaction was purchased from Perkin-Elmer and used as recommended. Preparation of C. reinhardtii chloroplast DNA for polymerase chain reaction reactions was as described by Berthold et al.(18) . Restriction fragments separated by agarose gel electrophoresis were transferred to nylon membrane (Amersham Corp.) as described by Southern(19) , and hybridization experiments were performed as described previously(20) . Fragments to be used as hybridization probes were separated by agarose gel electrophoresis and recovered from gel slices using Geneclean(TM) (BIO 101, Inc., La Jolla, CA). DNA fragments to be used as hybridization probes were radiolabeled with [alpha-P]dCTP by random-primed labeling(21) . Fluorograms of Southern blots were obtained on Kodak X-Omat AR(TM) x-ray film at -85 °C with Lightning Plus(TM) intensifying screens (DuPont NEN).

Construction of Strains DeltapetA and site-specific mutants Y1P, Y1F, Y1S, Y1W, P2V, and V3P

The 2.3-kilobase pair KpnI fragment containing the petA gene was subcloned from the chloroplast DNA BamHI restriction fragment 7 (nomenclature in Harris(22) ) into the KpnI site of pT7/T3alpha-19 (Life Technologies, Inc.) to generate plasmid pJB101. Deletion of the petA coding region was accomplished by digestion of pJB101 with HindIII and AflII, rendered blunt-end by treatment with Klenow fragment of DNA polymerase I, and self-ligation of the remaining plasmid to give plasmid pJB101-DeltaHAf (Fig. 1).

Figure 1: Restriction map of the petA gene in C. reinhardtii and the location of a deletion mutant of the petA gene. Ac = AccI, Af = AflII, B = BamHI, G = BglII, H = HindIII, K = KpnI, P = PstI.


To construct the N-terminal mutants by site-directed mutagenesis, the 0.8-kilobase pair HindIII-AflII fragment, containing most of the coding sequence of the petA gene, was subcloned from pJB101 into phage vector M13mp18 to give M13-petA, or the 2.3-kilobase pair KpnI fragment was subcloned into phagemid pTZ19 (Bio-Rad). Site-directed mutagenesis was performed by the method of Kunkel(23, 24) . M13-petA or pTZ19-petA single-stranded DNA was used for annealing with a mutagenic oligonucleotide (Table 1) and the mutant DNA strand was synthesized. The underlined nucleotide in Table 1indicated the mutagenic nucleotides. Mutant phage progenies were screened by DNA sequencing analysis. The HindIII-AflII fragment carrying the mutant sequence was then excised and re-cloned into plasmid pJB101 to replace the wild-type petA sequence to give the full-length of the mutated petA gene.

Chloroplast Transformation in C. reinhardtii

Chloroplast transformation in C. reinhardtii wild-type and DeltapetA strain was performed using biolistics technique following the procedures described by Boynton et al.(11) and Newman et al.(12) . Wild-type 7d or DeltapetA cells grown to approximately 3 times 10^6 cells/ml were harvested by centrifugation and resuspended in HS medium to a concentration of approximately 1 times 10^8 cells/ml. One-half milliliter of the cell suspension was then added to an equal volume of 0.4% agar (previously melted and kept at 42 °C), swirled, and spread on plates containing 1.5% agar in HS medium. The cells on the plates were bombarded with tungsten particles coated with appropriate donor DNA. The donor plasmid DNA (about 5 µg) was precipitated onto tungsten particles as described by Newman et al.(12) . For co-transformation experiments, the ratio of donor DNA to drug resistant marker DNA (in this experiment, the aadA cassette(25) ) was 4:1. The bombarded cells were incubated at 20 °C for 3 h under dim light and then transferred to HSHA plates containing 150 µg/ml of spectinomycin and incubated at 25 °C under dim light. The mutant strains were maintained on plates in HS medium under low light conditions.

Gel Electrophoresis, Heme Staining, and Immunoblotting

Protein and/or total cellular protein samples were separated on SDS-polyacrylamide gel electrophoresis with a 10-20% resolving gel and a 4% stacking gel, in a Laemmli buffer system(26) . Prior to electrophoresis, samples were solubilized in solubilization buffer containing 100 mM dithiothreitol, 1% SDS, 10 mM Tris-HCl (pH 8.3), and 5% glycerol, at 55 °C for 20 min. Following electrophoresis, heme-containing proteins were visualized by tetramethylbenzidine staining(27) . For immunoblot analysis, proteins separated on SDS-polyacrylamide gel electrophoresis were electroblotted onto nitrocellulose paper (Schleicher & Schuell) as described by Towbin et al.(28) . The transfer buffer contained 25 mM Tris (pH 8.3), 180 mM glycine, 20% methanol, and 0.1% SDS. The blocking solution contained 20 mM Tris (pH 7.5), 500 mM NaCl, and 2% (w/v) nonfat milk powder. C. reinhardtii cytochrome f was probed with a polyclonal antibody raised in rabbits against spinach cytochrome f and detected by ECL(TM) immunoblotting system (Amersham).

Oxygen Evolution Measurements

Chlorophyll concentrations were determined by the method of Arnon(29) . Oxygen evolution from whole cells was measured at 25 °C with a Clark-type electrode at a chlorophyll concentration of 13 µg/ml in 40 mM Hepes buffer, pH 7.5, plus 10 mM bicarbonate. Saturating light (1200 µE m s) was provided with a tungsten halogen light source with a 5-cm water bath as heat filter. Rates of light-dependent oxygen evolution were corrected for oxygen uptake arising from dark respiration.

Spectral Analysis of Cytochrome f

Chemical difference spectra of cytochrome f in membranes were measured in the presence of 1% Triton X-100 as described by Bendall et al.(30) and Bendall and Rolfe(31) . Membranes were prepared from whole cells by sonication and were isolated by differential centrifugation. A final chlorophyll concentration of 100-150 µg/ml was used for the assays. Hydroquinone minus ferricyanide difference spectra were recorded in a Shimadzu model UV-160A recording spectrophotometer.

Flash Kinetic Measurements

Kinetic measurements were made on permeabilized cell preparations (^1)to allow for easier redox poising than with intact cells. Wild-type or mutant cells of C. reinhardtii were grown autotrophically under high light conditions. Cells (1-2-liter cultures) were harvested at mid-exponential phase by centrifugation at 2500 times g for 6 min. The pellet was washed once with 400 ml of Buffer A (50 mM TES/KOH buffer (pH 7.2), 300 mM sorbitol, 1 mM MgCl(2), 1 mM MnCl(2), 2 mM EDTA, and 3 mM KH(2)PO(4)). The cells were then resuspended in Buffer A to a cell density of 1 times 10^8 cells/ml, passed through the Bionebulizer (32) at a pressure of 10 p.s.i., and then loaded onto the top of a Percoll step gradient that consisted of 5 ml of 55%, 7 ml of 40%, 7 ml of 30%, 7 ml of 20%, and 2 ml of 10% (v/v) Percoll prepared in Buffer A in a 45-ml centrifuge tube. The gradients were centrifuged in a Beckman JS-13.1 swinging bucket rotor at 10,000 times g for 10 min. After centrifugation, the band at the interphase between the 40% and the 30% Percoll layers was collected, diluted 10-fold with Buffer A, and centrifuged at 2500 times g for 4 min. The pellet was resuspended with the same buffer to a final chlorophyll concentration of approximately 1 mg/ml and used for spectroscopic measurements. Microscopic examination of this fraction showed that >90% of the nebulized cells have a continuous border region and distinct cup-shaped chloroplasts but no flagella. In contrast with intact cells, this nebulized cell fraction is not refringent in Buffer A when observed with phase contrast. We believe that this fraction represents permeabilized cells that have retained much of the soluble components of the chloroplasts. For example, we have found that plastocyanin, the electron donor to Photosystem I, is retained and that the electron transport pathway from Photosystem II to ferredoxin is fully functional (data not shown).

Flash-induced absorbance changes were measured at 25 °C under anaerobic conditions maintained by argon flux. Nebulized cells were suspended (to a final chlorophyll concentration of 30 µg/ml) in 10 mM MOPS/KOH buffer (pH 7.0), 20 mM KCl, and 300 mM sorbitol. The following redox mediators (10 µM each) were present: 1,2-naphthoquinone; 1,4-naphthoquinone; p-benzoquinone; 2,5-di-OH-p-benzoquinone; juglone; and riboflavin-5-monophosphate. The preparation was uncoupled by the addition of 10 µM of valinomycin, nigericin, and gramicidin. Redox potentials were adjusted (with dithionite or ferricyanide) and monitored as in (33) . Cytochrome f was monitored at 554-545 nm and P700 (^2)at 702-730 nm using a spectral bandwidth of 2.9 nm. Extinction coefficients of 20 for cytochrome f and 64 for P700 were used(34, 35) . A home-built single-beam kinetic spectrophotometer with microsecond time resolution was used. In order to minimize actinic effects from the measuring beam, a shutter was placed before the sample and was opened 200 ms prior to the flash activation. This was provided by two FX201 EG& flashlamps through a red RG 695 Schott filter. The measured combined flash duration was 3.5 µs at half-peak height. The photomultiplier was protected by a blue-green filter, while for P700 measurements the filters were interchanged. For both cases the flash saturation was approximately 90%. Dark time between repetitions during averaging was 40 s. Data were acquired with a Nicolet model 206 transient recorder and then transferred to a computer for subsequent averaging and analysis. Further details of this instrument will be described in a subsequent publication. A program written by Dr. Ed Berry of the Lawrence Berkeley Laboratory (Berkeley, CA) was used for spectral deconvolution.

To test that the pair of wavelengths (554-545 nm) was specifically monitoring cytochrome f, we compared the observed signal with the one provided by a full spectral deconvolution (34) at the defined oxidation-reduction potentials of 390, 160, and 0 mV. The results from both approaches are almost identical as regards the extent and kinetics of the cytochrome for a train of eight flashes 60 ms apart. This demonstrates that the wavelength pair 554-545 nm satisfactorily deconvolutes cytochrome f from other overlapping signals at a variety of redox change levels of cytochrome f and of the other components of the electron transfer chain.

RESULTS

Molecular Characterization of C. reinhardtii Cytochrome f Mutants

The studies in this work were facilitated by the preparation of a C. reinhardtii mutant, known as DeltapetA, which had a deletion in the coding region for petA, the chloroplast gene encoding cytochrome f. A detailed description of the phenotype of this strain will be presented in another report, (^3)but, as expected it did not grow photoautotrophically in either high or low light due to the complete absence of cytochrome f. Other components of the cytochrome b(6)f complex were also markedly reduced in the deletion mutant. A series of N-terminal mutants in the cytochrome f protein was prepared by site-directed mutagenesis followed by chloroplast transformation of the mutated petA gene into the DeltapetA strain of C. reinhardtii. The mutants that have been prepared are identified as the following: Y1P, Y1F, Y1S, Y1W, P2V, and V3P, as shown in Table 1. A reconstructed wild-type strain was also prepared by transformation of the wild-type petA gene into the DeltapetA strain to serve as a control, and this reconstructed wild-type strain (wild-type) was used for comparison in all experiments described in this report. The correct integration of the mutated petA gene into its chloroplast genome locus was examined by Southern blot analysis and, as shown on the fluorogram, all of the mutant cytochrome f transformants contain the wild-type length of the petA specific DNA band which indicated proper integration of the transformed cytochrome f DNA into its chloroplast genome locus has occurred (Fig. 2). The presence of the mutant DNA sequence in these transformants was confirmed by DNA sequencing analysis using polymerase chain reaction-amplified DNA templates from the respective mutant strain (data not shown). The presence of cytochrome f in the strains was analyzed by immunoblotting (Fig. 3), which identifies the apoprotein of cytochrome f in all strains except the deletion strain and the Y1P mutant, and by heme-staining (data not shown), which identifies the holoprotein. Except for these last two, all of the strains showed photoautotrophic growth with the rates varying depending on the light intensity (see below).

Figure 2: Southern blot hydridization of C. reinhardtii wild-type and cytochrome f mutant DNAs. Total DNA was isolated from wild-type and cytochrome f mutant strains and digested by BglII/PstI. A 1.9-kilobase pair BglII/KpnI fragment containing the entire petA coding region plus its 5`,3`-flanking sequence (see Fig. 1) was used as a probe for the hybridization. Lanes 1-8 contained DNAs from wild-type, DeltapetA, Y1P, Y1F, Y1S, Y1W, P2V, and V3P, respectively.


Figure 3: Immunoblot analysis of the cytochrome f protein in wild-type and mutant cells of C. reinhardtii. Total cellular protein from C. reinhardtii wild-type, DeltapetA, Y1P, Y1F, Y1S, Y1W, P2V, and V3P (lanes 1-8) was isolated, electrophoresed, and reacted with an antibody raised against spinach cytochrome f, as described under ``Materials and Methods.'' Amounts of sample loaded in each lane were not equivalent.


Electron Transfer Chain Stoichiometries

Results with several preparations for each strain show that they have the same photosystem I unit size (660 ± 30 chlorophyll a+b/P700). The preparations are also similar in the extent of their fast re-reduction of P700 (submillisecond kinetics), measured 3 ms after flash activation (instrument time constant = 200 µs), which in fresh samples is 70-80% of the maximum photooxidizable P700. In samples aged in the cuvette for 60-90 min, the extent of P700 fast re-reduction decreases to 65%, presumably due to a loss of plastocyanin. The residual phase(s) have an average t 20 ms. The fast phase indicates the presence of kinetically competent plastocyanin, which is able to reduce the photosystem I reaction center in the microsecond time domain (see (36) ).

Table 2shows that comparable amounts of photooxidizable cytochrome f are detected in all strains, except that V3P and P2V have a slightly higher level (25-20%) than the wild-type strain. The partial instability of the preparations in relation to their plastocyanin level can explain the variability in the contents of photooxidizable cytochrome f in different preparations of the same strain and, at least in part, the differences shown in the cytochrome f/P700 ratio in the V3P and P2V strains. Indeed, the above-described decrease of the fraction of P700 showing rapid re-reduction is accompanied by a decrease in the level of the cytochrome f signal. Such a decrease could be avoided by maintaining the sample for not more than 90 min. This instability points to the need of normalizing the observed stoichiometries by a factor of 1.4 in order to calculate the actual stoichiometry of the cytochrome b(6)f complex/P700 in vivo (data in Table 2are not normalized). As the wild-type and all mutant strains have the same content of chlorophyll a+b/cell (1.4 ± 0.1 pg/cell), we conclude that the contents of cytochrome b(6)f complex per cell are also similar.

Kinetics of Cytochrome f Photooxidation in C. reinhardtii petA Mutants

Measurements of the kinetics of cytochrome f photooxidation were carried out in samples poised at an E(h) of approximately 0 mV. At this potential, the high potential electron carriers, including P700, plastocyanin, cytochrome f, and the Rieske iron-sulfur protein, were in the reduced state prior to flash activation. The quinone pool would also be in the reduced state in the dark, while the b cytochromes would be predominantly oxidized.

It has been reported by Delosme (36) that cytochrome f in C. reinhardtii shows complex oxidation kinetics after flash activation, with reported t from 80 to 500 µs that were dependent on physiological factors. As shown in Fig. 4, we find in permeabilized cells of the wild-type C. reinhardtii that, after single flash activation, cytochrome f photooxidation occurs with an apparent t of 250-350 µs, using an instrument time constant of 20 µs. Also shown in this figure are the kinetics of cytochrome f oxidation for one of the N-terminal mutants, P2V, and within the experimental error in these kinetic traces, there is no substantial difference in oxidation rates as compared with the wild-type strain. The t values of oxidation of all the N-terminal cytochrome f mutants are summarized in Table 2, and the results show little effect on oxidation rates induced by these mutations.

Figure 4: Kinetics of cytochrome f oxidation in wild-type and the petA-P2V mutant of C. reinhardtii. Flash-induced absorbance changes were measured in permeabilized cells as described under ``Materials and Methods'' in the presence of 2 µM stigmatellin at an E(h) of 0 ± 10 mV and instrument time constant of 20 µs. Traces shown are the result of 160 averages using several preparations.


Kinetics of Cytochrome f Re-reduction in C. reinhardtii petA Mutants

The kinetics of cytochrome f photooxidation and re-reduction following a single flash in the wild-type strain of C. reinhardtii are shown in Fig. 5A. Because of the time scale used in these traces, the oxidation kinetics are not resolved, but the reduction kinetics can be shown to occur in the millisecond time range. In order to adequately resolve the kinetics of cytochrome f re-reduction, the specific inhibitor of the cytochrome b(6)f complex, stigmatellin, was used. Stigmatellin is known to inhibit the oxidation of quinol by cytochrome b(6)f and bc(1) complexes at the quinol oxidation site (Q(z), Q(o) or Q(p)) by raising the midpoint potential of the Rieske iron-sulfur center, thereby impairing its ability to reduce the heme group of cytochrome f(37, 38) . As shown in Fig. 5B, the observable extent of cytochrome f oxidation is increased in the presence of stigmatellin due to the inhibition in the rate of re-reduction. The slow reduction of the cytochrome in the presence of stigmatellin presumably originates from redox mediators present in the reaction mixture and/or partial leakage of the inhibitor. A similar analysis of petA-P2V is shown in Fig. 6with the kinetics being measured in the absence of stigmatellin in Fig. 6A and in the presence of stigmatellin in Fig. 6B. In the absence of inhibitors, only 73% of the cytochrome f that is photooxidized with one flash in the presence of stigmatellin is re-reduced in 200 ms. The re-reduction phase (t = 43 ms) is monotonic for four to five t values. In different preparations, the extent of the re-reduction phase varies between 68 to 80%. On the contrary, in the wild-type Y1W, Y1F, Y1S, and V3P strains, a more complete re-reduction (in the 90-100% range) is observed in less than 60 ms. A lowering of the E(h) to -80 mV does not change the observed kinetics in the wild-type or P2V strain, giving confidence that the PQ pool was fully reduced in all cases.

Figure 5: Kinetics of cytochrome f re-reduction in wild-type C. reinhardtii. Flash-induced absorbance changes were measured in permeabilized cells as described under ``Materials and Methods.'' A, no additions. B, plus stigmatellin (2 µM). The E(h) was adjusted to 0 ± 10 mV, and an instrument time constant of 100 µs was used. Traces shown are the result of 80 averages using the same preparation.


Figure 6: Kinetics of cytochrome f re-reduction in the petA-P2V mutant of C. reinhardtii. Flash-induced absorbance changes were measured in permeabilized cells as described under ``Materials and Methods.'' A, no additions. B, plus stigmatellin (2 µM). The E(h) was adjusted to 0 ± 10 mV and an instrument time constant of 500 µs was used. Traces shown are the result of 40 averages using the same preparation.


To assess the rate of cytochrome f re-reduction by electrons passing specifically through the Rieske iron-sulfur center, the kinetics of cytochrome f re-reduction sensitive to stigmatellin were evaluated by subtraction of the traces in the presence of stigmatellin from those in the absence of the inhibitor. These results for all strains are summarized in Fig. 7. It is shown that cytochrome f re-reduction in the wild-type strain occurs with a t of 3 ms, whereas in the case of the P2V mutant, the rate of re-reduction has decreased to approximately 40 ms. This is the largest effect seen in any of the petA mutants that have been analyzed. For all strains and replicates, the stigmatellin-sensitive re-reduction of cytochrome f fits a single exponential for three t values. The extent of the stigmatellin-sensitive re-reduction in the P2V mutant appears smaller than in the other strains. However, this can be explained by the slow rate of re-reduction in the absence of inhibitors being partially mimicked in the inhibited condition by the slow nonspecific re-reduction via redox mediators and inhibitor leakage. Both phenomena would be expected to diminish the extent of re-reduction that is sensitive to stigmatellin and also to distort this signal at longer times after the flash.

Figure 7: Kinetics of cytochrome f re-reduction that is sensitive to stigmatellin in wild-type and mutants of C. reinhardtii. Flash-induced absorbance changes were measured in permeabilized cells as described under ``Materials and Methods.'' The differences between the traces in the absence and presence of 2 µM stigmatellin are shown. The E(h) was adjusted to 0 ± 10 mV, and an instrument time constant of 500 and 100 µs was used for the measurements in P2V and all the other strains, respectively. The traces are the average of 40-80 measurements for each preparation. The dotted line indicates the time of the firing of the flash. The traces for the wild type (wt) and P2V are the difference obtained from the data shown in Fig. 5and 6, respectively.


A summary of the results for the N-terminal mutants showing the t for re-reduction is given in Table 2. They show that mutants at the Tyr^1 position are only moderately affected, with petA-Y1S having the largest effect although even in this strain, the re-reduction rate has slowed to only 7.5-9.5 ms, while the other mutants, such as petA-Y1W and petA-Y1F, are only marginally affected (3-5 ms). Our results also indicate that a mutation in position 3, petA-V3P, slightly increases the rate of reduction of cytochrome f to 2.3 ms. As noted previously, the P2V mutant has a considerably slower rate of re-reduction of cytochrome f, in the 40 ± 5 ms range, if measured as the difference between ``no inhibitor minus stigmatellin traces'' (Fig. 7), and 60-75 ms, if measured directly from the relaxation after photooxidation in the absence of inhibitor (Fig. 6A) and not considering that the reduction is incomplete.

Besides slowing the cytochrome f re-reduction kinetics, the P2V mutation significantly affects the fraction of cytochrome f that is photooxidizable with the first flash in the presence of stigmatellin. Table 2shows that for all strains but P2V, the ratio of cytochrome f photooxidized in the first flash to the maximal photooxidation attainable in a train of flashes, ranges from 50 to 70% with, probably, no significant differences. In contrast, for P2V, this value is 75-90%. This could be explained if the cytochrome f heme group in P2V has a lower E(m) value than in the wild-type strain (where E(m) is the midpoint redox potential), displacing the reaction toward a more complete re-reduction of plastocyanin. We note, however, that there is no change in the oxidation kinetics of cytochrome f in the P2V mutant, although the reduction kinetics are altered.

Spectral Analysis of Cytochrome f

Because of the unusual kinetic features of cytochrome f in the P2V mutant, we measured chemical difference spectra of cytochrome f in Chlamydomonas thylakoid membranes to determine if there were any substantial changes in the spectrum of the cytochrome. This analysis was done by measuring the hydroquinone minus ferricyanide difference spectrum in the presence of 1% Triton X-100, which is used to convert cytochrome b to its low potential form and remove its spectral overlap with cytochrome f(30, 31) . This analysis showed the wild-type and P2V strains had approximately the same content of cytochrome f in their membranes on a chlorophyll basis (approximately 1 cytochrome f/500-600 chlorophylls), that this cytochrome f was hydroquinone-reducible in both cases, and that all spectra had an absorbance peak at 554 nm (data not shown). This value for the stoichiometry and the absorbance peak of the cytochrome agrees with the estimation based on photooxidizable cytochrome f in light-induced measurements.

Growth Rates and Oxygen Evolution of Whole Cells of petA Mutants of C. reinhardtii

The rate of photoautotrophic growth of the petA mutants was measured in a minimal medium that contained CO(2). As shown in Table 3, all of the mutants had comparable growth rates at low light intensities (approximately 35 µE m s), but differences became apparent at saturating light intensities (approximately 350 µE m s). Under the high light intensities, the growth rate of the petA-P2V strain was approximately 60% of that of the wild-type strain. Other mutants, such as petA-Y1W or petA-V3P, showed no changes in growth rate at high light intensities, while other position 1 mutants, petA-Y1F and petA-Y1S, had slightly inhibited growth rates as compared with the wild-type strain. The results at both light intensities for the wild-type and P2V strains were also confirmed by cell counting.

Whole cell rates of oxygen evolution for the mutants are also shown in Table 3. The largest effect is with the P2V mutant where a 33% inhibition was observed. Other strains, such as Y1F and Y1S, showed a slight inhibition of approximately 20%.

DISCUSSION

The recent structural analysis of turnip cytochrome f revealed an unusual ligation of the c-type heme covalently bound in this protein when the N-terminal amino tyrosine residue was found to provide the sixth ligand to the iron via its alpha amino group (8) . The position of the aromatic ring of this tyrosine residue, lying parallel to the heme plane, suggests a role in the transfer of electrons into the heme group. We have tested this idea directly using site-directed mutagenesis of the N-terminal tyrosine. In the case of the replacement of this residue with a proline, which does not contain a primary amine that can serve as a sixth heme ligand, we find that the Chlamydomonas mutant is unable to grow photosynthetically and that there is no cytochrome f and other components of the cytochrome b(6)f complex present in this organism. This result is similar to those of Kuras and Wollman(39) . For mutants in which cytochrome f is not assembled, there is no photoautotrophic growth of the strain and all components of the cytochrome b(6)f complex are absent. This result suggests that the Chlamydomonas cytochrome b(6)f complex assembles by an ``all-or-none'' mechanism, and in this respect, this complex differs from the analogous cytochrome bc(1) complex (40, 41, 42) . Similar conclusions have been put forward in studies of a nuclear Lemna mutant deficient in all of the components of the cytochrome b(6)f complex (43) .

In the cases in which the N-terminal tyrosine residue has been replaced by other aromatic amino acid side chains, such as Phe or Trp, the effects on the function of cytochrome f are minimal. All of these mutants assemble a functional cytochrome f and grow photoautotrophically. These mutant strains show kinetics of cytochrome f photooxidation and re-reduction that are only marginally altered in comparison with the wild-type strain. Rates of growth of these mutants are also similar to wild-type rates. It should be noted that in the case of Marchantia polymorpha, the N-terminal amino acid of cytochrome f is Phe, indicating that a natural replacement with another aromatic group can occur(44) . In the case of the replacement of Tyr^1 with the nonaromatic, polar serine residue, we find a somewhat larger effect on the re-reduction kinetics, but there is still only a rather small effect on growth. We conclude from these studies that, whereas the presence of the N-terminal primary amine is essential for the assembly of cytochrome f, an N-terminal aromatic side chain has no critical role in the transfer of electrons into or out of the heme group. These results should be contrasted with the recent work on cytochrome c(1) of Rhodopseudomonas capsulatus where modification of the presumed axial methionine ligand to the heme, M183 in this organism, resulted in a loss of photosynthetic growth and large change in the midpoint potential of the mutated cytochrome(45) .

In the site-directed cytochrome f mutant where Pro^2 has been replaced with a Val residue, more dramatic effects on the function of cytochrome f have been observed. In 13 different organisms where complete sequences of cytochrome f are available, Pro^2 is conserved, indicating an important function in the protein(6) . In our P2V mutant, while the rate of photooxidation of the cytochrome is unaffected, the rate of re-reduction is decreased by over a factor of 10, from t of 2.5-3.5 ms to approximately 40 ms. This is the largest effect that we have observed for any of the N-terminal region mutants studied in this work. Pro^2 forms part of the front face of the pocket into which the heme is inserted. It is somewhat surprising that changes in this amino acid has a greater effect on cytochrome f than do changes in the N-terminal amino acid, but because of the unusual features of proline in terms of disrupting helices and producing ``kinks'' in peptide chains, our findings are not totally unanticipated. It is also of note that the single mutation made in the third position, a conversion of valine to proline, yielded a mutant protein that actually showed faster reduction rates for cytochrome f than the wild-type strain, although there was no effect on the growth rate. On the basis of these results, we would propose that a disruption of the helical structure near the N terminus of cytochrome f (residues 2 or 3) contributes to an adequate configuration for electron transfer from the Rieske center to the heme group of the cytochrome.

It is important to emphasize that the slow re-reduction kinetics observed in mutants such as Y1S and P2V are an intrinsic effect at cytochrome f due to the single mutations. The mutants do not exhibit either a higher stoichiometry of light-induced oxidizing equivalents or a smaller pool of interconnecting plastocyanin in a system with delocalization of the high potential chain(46) . These conditions could generate multiple turnovers of oxidation of the cytochrome f heme (the first one fast and the subsequent slower) after a single flash that could account for a slow overall re-reduction. Thus, the observed stoichiometry of photoactive cytochrome f/P700, although smaller than 1, is very similar for all strains. Second, the re-reduction of P700 is almost identical in all strains, showing that the interconnecting pool of plastocyanin is equivalent. Third, in Y1S and P2V the percent of the maximum of cytochrome f that can be photooxidized with the first flash in the presence of stigmatellin is larger and as fast and not smaller and slower than in the wild-type. The opposite would be expected if a lower ratio of plastocyanin/cytochrome b(6)f complex would be present in these mutants that have, nevertheless, similar ratios of cytochrome b(6)f/P700 as in the wild-type. Finally, a change in the level of actinic flash intensity in studies with the P2V mutant, causing a 3-fold decrease in the extent of photooxidation after one flash, does not change the slow kinetics of re-reduction (t 40 ms). As this excitation condition will minimize any excess of oxidation equivalents(47) , there is no coexistence of multiple turnovers of oxidation causing what would be an apparent slowing of the rate of re-reduction.

Taking into account that the mutants studied in this work affect residues that are very close to the heme, the unchanged rate of photooxidation suggests that electrons have different routes for equilibration with plastocyanin and the Rieske iron-sulfur center. While most of the mutations studied had little effect on the kinetics of cytochrome f as well as on photoautotrophic growth, the petA-P2V mutant showed a large decrease in growth under saturating light conditions. In contrast, at lower light intensities, the wild-type and all mutants, including P2V, grew photoautotrophically at the same rate, which is four times slower than the wild-type strain at saturating light intensities. These results indicate that, on the one hand, at low light growing conditions the limiting step for all strains is at the level of the photosystem(s) light activation, whereas at high light intensities in the P2V mutant the reactions of the cytochrome b(6)f complex, and, in particular, the reduction of cytochrome f via the Rieske iron-sulfur center (t = 40 ms), has become the rate-limiting step in the overall photosynthetic and growth process (see below). These conclusions are based on the fact that all the strains studied contain comparable amounts of cytochrome complex per photosystem I and per cell. In this context, it is interesting that in a mutant where electron flow is impaired, such as P2V, there is no increased synthesis of the cytochrome complex to compensate for the kinetic limitation at the cytochrome complex.

Through the behavior of the wild-type and mutant strains (V3P, Y1W, Y1F, and Y1S), encompassing a range of cytochrome f re-reduction t values from 1.5 to almost 10 ms, we have shown that under high light conditions, the turnover of the cytochrome f re-reduction when the plastoquinone pool is fully reduced is not the rate-limiting step for growth. In the wild-type, the rate of photoreduction of cytochrome b at an E(h) of 0 mV measured in the presence of 2-n-heptyl-4-hydroxyquinoline-N-oxide is very similar to the rate of the stigmatellin-sensitive re-reduction of cytochrome f (i.e. t of 2.5-3.5 ms), whereas its reoxidation, sensitive to the inhibitor, is slower (t of 15 ± 3 ms). (^4)Therefore it is impossible to determine here if a cytochrome b(6)f complex with an overall rate-limiting step of t 10-15 ms in the strains (including the wild-type) is not the limiting step for growth. Other candidates for metabolic limiting steps could be at the level of CO(2) fixation or subsequent anabolic reactions. We interpret the nonlinearity of the oxygen evolution rates with the cytochrome f stigmatellin-sensitive re-reduction rate in a similar fashion. In contrast, in the P2V mutant, the rate of re-reduction of cytochrome f has clearly become the limiting reaction in photosynthesis under light-saturating conditions such that a reduction in this rate, due to a single mutation, alters the growth rates. Whether the mutated cytochrome b(6)f complex in the P2V mutant causes inhibition of growth and oxygen evolution only by limiting the rate of electron flow or also by generating a pleiotropic effect, such as enhanced photoinhibition stress due to slow reoxidation of plastoquinol (see (48) ), remains to be determined.

FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant GM-20571 (to R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Plant Biology, 111 Koshland Hall, University of California, Berkeley, CA 94720-3102. Tel.: 510-642-5959; Fax: 510-642-4995; dickm{at}nature.berkeley.edu.

(^1)
J. Fernandez-Velasco and R. Malkin, manuscript in preparation.

(^2)
The abbreviation used is: P700, photosystem I reaction center chlorophyll; E, einstein.

(^3)
J. Zhou, J. Fernandez-Velasco, and R. Malkin, manuscript in preparation.

(^4)
J. Fernandez-Velasco, J. Zhou, and R. Malkin, manuscript in preparation.

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