(Received for publication, November 17, 1995; and in revised form, January 9, 1996)
From the
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 bf 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
= 0 mV, (where E
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.
The cytochrome bf 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
complex, and this
analogy extends to their composition(1, 4) . Among the
electron transfer carriers in the cytochrome b
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 -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
-amino group of
Tyr
, 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
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
, Phe
, Pro
, and
Pro
, all of which form the hydrophobic pocket.
We have
been studying structure-function relationships in the cytochrome bf 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
and
Val
have been examined. We find that mutants in the
Tyr
position show comparatively small effects in terms of
electron transport and growth, while a Pro
mutant is
markedly affected. These results are discussed in terms of structural
and functional aspects of the cytochrome f molecule.
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.
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 ()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.
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, petA, 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, petA, 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.
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
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
f complex per
cell are also similar.
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 of 0 ± 10 mV and instrument time constant
of 20 µs. Traces shown are the result of 160 averages using several
preparations.
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 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 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 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
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 value than in the wild-type strain (where E
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.
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%.
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 bf 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
f complex
are absent. This result suggests that the Chlamydomonas cytochrome b
f complex assembles by
an ``all-or-none'' mechanism, and in this respect, this
complex differs from the analogous cytochrome bc
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
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 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
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 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
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
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 bf complex would
be present in these mutants that have, nevertheless, similar ratios of
cytochrome b
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 bf 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
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). (
)Therefore it is impossible to
determine here if a cytochrome b
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
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
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.