Functional Insensitivity of the Cytochrome b6 f Complex to Structure Changes in the Hinge Region of the Rieske Iron-Sulfur Protein*
Jiusheng Yan and
William A. Cramer
From the
Department of Biological Sciences, Purdue University, West Lafayette,
Indiana 47907-2054
Received for publication, December 11, 2002
, and in revised form, March 13, 2003.
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ABSTRACT
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Structure analysis of the cytochrome bc1 complex in the
presence and absence of Qp quinol analog inhibitors implied that a
large amplitude motion of the Rieske iron-sulfur protein (ISP) is required to
mediate electron transfer from ubiquinol to cytochrome c1.
Studies of the functional consequences of mutagenesis of an 8-residue ISP
"hinge" region in the bc1 complex showed it to
be sensitive to structure perturbation, implying that optimum flexibility and
length are required for the large amplitude motion. Mutagenesis-function
analysis carried out on the ISP hinge region of the cytochrome
b6 f complex using the cyanobacterium
Synechococcus sp. PCC 7002 showed the following. (i) Of three
petC genes, only that in the petCA operon codes for
functional ISP. (ii) The function of the complex was insensitive to changes in
the hinge region that increased flexibility, decreased flexibility by
substitutions of 46 Pro residues, shortened the hinge by a 1-residue
deletion, or elongated it by insertion of 4 residues. The latter change
increased sensitivity to Qp inhibitors, whereas deletion of 2
residues resulted in a loss of inhibitor sensitivity and a decrease in
activity, indicating a minimum hinge length of 7 residues required for optimum
binding of ISP at the Qp site. Thus, in contrast to the
bc1 complex, the function of the b6
f complex was insensitive to sequence changes in the ISP hinge that
altered its length or flexibility. This implies that either the barriers to
motion or the amplitude of ISP motion required for function is smaller than in
the bc1 complex.
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INTRODUCTION
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The cytochrome b6 f complex functions as a
plastoquinol: plastocyanin/cytochrome c6 oxidoreductase in
oxygenic photosynthesis and is shared by both photosynthetic and respiratory
electron transfer pathways in cyanobacteria
(1). It mediates electron
transfer from photosystem II to photosystem I in the linear electron transfer
pathway or around photosystem I in a cyclic pathway, generating an
electrochemical proton gradient across the membrane that is used for the
synthesis of ATP (2,
3). The b6
f complex is phylogenetically analogous to the cytochrome
bc1 complex of mitochondria and photosynthetic bacteria.
It is an integral membrane protein complex composed of eight to nine
polypeptide subunits (3,
4), the four largest (>17
kDa) of which have defined functions. The 24-kDa cytochrome
b6 subunit, which has four transmembrane
-helices
and contains two b-type hemes, together with the 17-kDa subunit IV, which has
three transmembrane helices, are homologous to the N- and C-terminal segments
of the cytochrome b in the bc1 complex
(5). The 19-kDa Rieske
iron-sulfur protein
(ISP),1 consisting of
an N-terminal single transmembrane
-helix domain and a 140-residue
soluble extrinsic domain with a linker region connecting these two domains,
has an overall function similar to that of the ISP in the
bc1 complex, deprotonating the membrane-bound quinol and
transferring electrons from the quinol to the membrane-bound c-type
cytochrome (6,
7,
8). Of the two subdomains of
the ISP extrinsic domain, the cluster-binding small subdomain involved in
formation of the Qp site (the quinone-binding site on the
electrochemically positive side of the b6
f/bc1 complex) is structurally identical in the
two complexes, whereas the large subdomain, which is directly connected to the
linker region, has a different fold
(9). In addition, the structure
of the membrane-bound c-type cytochrome is completely different in
the two complexes. The 31-kDa c-type cytochrome f subunit is
functionally related to, but structurally completely different from, the
cytochrome c1 in the bc1 complex
(10,
11). The structure
differences, as well as the similarities, between these two complexes are
important for understanding the mechanism of the ISP in the
b6 f complex (see below).
High resolution x-ray diffraction analysis of the mitochondrial cytochrome
bc1 complexes in the presence/absence of the Qp
site inhibitor stigmatellin suggested an intracomplex electron shuttle
function for the ISP subunit, which mediates electron transfer from ubiquinol
at the Qp site to cytochrome c1 through a
60° rotation of its extrinsic soluble domain and a resulting 16-Å
displacement of the [2Fe-2S] cluster (Fig.
1) (7,
12,
13,
14,
15,
16). In the crystal
structures, the displacement of the ISP extrinsic domain is accompanied by a
large conformational change in the hinge region (flexible part of the linker
region), whereas the internal structure of the extrinsic domain and the
position of the membrane anchor domain are little changed at
3-Å
resolution (Fig. 1)
(12,
14). It was subsequently found
that the function of the complex is very sensitive to perturbation of the
sequence and structure of the hinge region by site-directed mutagenesis in
which the hinge was altered by residue deletion, insertion, and substitution
(17,
18,
19,
20,
21,
22,
23,
24,
25,
26). Although these data were
interpreted in terms of a requirement for the mobility of the ISP soluble
domain, the extreme sensitivity of bc1 function to
increases in hinge flexibility
(19) and length
(17,
24,
26) cannot be predicted or
inferred from the x-ray structure data. Thus, it is of interest to study the
function of the putative hinge region in the b6 f
complex (Table I).

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FIG. 1. The two extreme positions of the ISP extrinsic domain and the
corresponding conformational changes in the hinge region of the avian
cytochrome bc1 complex
(12). The ISP located at
the b position is shown in blue, and that at the
c1 position in green. Left, two different
positions of ISP in the context of the cytochrome (cyt) b
subunit and cytochrome c1 heme; right, artificial
superposition of the ISP extrinsic domain at its two extreme positions, to
demonstrate that its motion involves a 60° rotation. Structures were
drawn with MOLSCRIPT (49) and
RASTER3D (50).
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TABLE I Sequence alignment of the ISP hinge region in the cytochrome
bc1 and b6f
complexes Asterisks indicate invariant residues; colons indicate highly
conserved residues; and periods indicate conserved residues.
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Because the structure of the ISP and the predicted structures of the
cytochrome b6 and subunit IV in the b6
f complex are similar to the structures of the ISP and cytochrome
b in the bc1 complex, it is expected that the ISP
in the stigmatellin- or DBMIB-bound b6 f complex
is in a position and orientation similar to those observed in the
stigmatellin-bound bc1 complex
(Fig. 2). Three lines of
evidence indicate that the ISP in the b6 f
complex may also act as a mobile redox center. (i) EPR studies defined
different orientations for the [2Fe-2S] cluster of the ISP extrinsic domain in
the presence and absence of the Qp site quinol analog inhibitor
DBMIB (27) or inhibitory metal
ions (28). (ii) The rate of
flash-induced reduction of cytochromes b6 and f
is systematically inhibited as a function of ambient extrinsic viscosity
in situ (29). (iii)
An 8-residue segment containing 47 Gly, 03 Ser, 01 Thr,
and 02 Ala residues, whose alignment with the set of Rieske ISP
sequences suggests that it is a putative hinge region, is present in the
b6 f complex
(Table I). The sequence of this
segment is conserved in the ISP sequences of b6 f
complexes from various organisms (Table
I) and suggests a higher degree of conformational flexibility than
in the bc1 complex. Thus, it is anticipated that a
movement of the ISP soluble domain is involved in electron transfer from
quinol to cytochrome f in the b6 f
complex. To probe the role of the putative ISP hinge region in the
b6 f complex, the cyanobacterium
Synechococcus sp. PCC 7002, previously used for site-directed
mutagenesis of cytochrome b6 and subunit IV
(30), was used for mutagenesis
of the ISP. In contrast to the bc1 complex, the function
of the b6 f complex was found to be insensitive
to large perturbations of the ISP hinge region, implying major differences
between the complexes either in structural barriers to motion of the ISP
soluble domain or in the amplitude of its motion.

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FIG. 2. Interaction of the ISP soluble domain with the p side of
cytochrome b in the stigmatellin-bound avian bc1
complex (12) (A) and
of cytochrome b6 and subunit IV in the modeled structure
of the b6 f complex (B). The contact
between the 23-strand region (site a) of the
ISP and the ef loop (site b) of cytochrome (cyt)
b in the bc1 complex or between cytochrome
b6 and subunit (su) IV in the
b6 f complex is labeled as a fulcrum region and
highlighted by a blue circle. Structures were drawn with MOLSCRIPT
(49) and RASTER3D
(50).
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MATERIALS AND METHODS
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Growth of CulturesSynechococcus sp. PCC 7002 wild-type and
mutant strains were grown in medium A
(31) supplemented with
spectinomycin at 100 µg/ml for mutants. Liquid cultures were grown at 38
°C under cool-white fluorescent illumination at a light intensity of
100 microeinsteins/m2/s, bubbled with air supplemented with
2.0% (v/v) CO2. Cell densities were determined from the
absorbance at 730 nm.
Site-directed Mutagenesis and Plasmid ConstructionA
PCR-based site-directed mutagenesis method (Stratagene) was performed as
described (32). The presence
of mutations and the intactness of the remainder of the petC1 genes
on plasmids and in the Synechococcus genome were analyzed by DNA
sequencing. For mutants 4Pro
[PDB]
, 6Pro, and +4G, the intactness of their genomic
petB and petD genes, which encode cytochrome
b6 and subunit IV, was confirmed by sequencing. A 3.5-kb
segment encompassing the petCA operon and 1.5 kb of the
5'-flanking sequence was digested from pUHWRW1
(33) with XbaI and
KpnI and cloned into the XbaI and KpnI sites of
pUC19 to generate plasmid I. A HindIII site (silent mutation) was
introduced into the 3'-end of the petC1 gene by site-directed
mutagenesis with CTG TCC AGT CGG TGA AGC TTC ACC GAC TGG ACA G
(mutation sites are underlined) and its complementary primer to generate
plasmid II, which was digested with HindIII; and the resulting 4.5-kb
segment was cyclized by ligation to generate plasmid III. A new PacI
site was introduced at a site
30 bp upstream of the petC1 start
codon by mutagenesis with GGA AGT ATT GTA GAA ACG CTT AAT TAA CTA
TAC AAA AGG CTC TG and its complementary primer. The resulting plasmid IV was
linearized at a site
500 bp upstream of petC1 by PCR with
Pfu polymerase and primers GGC CCG TTC TCC CAA TTC C and GGC AGA ATG
GCT GTT GGG TTA ATC C. The aadA cassette
(34), which encodes an
aminoglycoside 3'-adenyltransferase and confers spectinomycin resistance
to the host strains, was ligated with the linearized plasmid IV to generate
plasmid V, which contains an aadA cassette located
500 bp
upstream of petC1. A 3.8-kb fragment including the 5'-part of
petC1 and the 5'-flanking sequence with the aadA
cassette was excised from plasmid V by restriction digestion with
PmlI and BamHI and ligated into the PmlI and
BamHI sites of plasmid I to generate plasmid pCA1, which contains the
whole petCA operon and an inserted aadA cassette (2.0 kb) in
its 5'-flanking sequence (1.5 kb). This was the final plasmid used to
transform the Synechococcus cells. pCA1 was digested with
PmlI and HpaI to remove the 5'-flanking sequence and
then religated to create construct pCA2. Mutations in petC1 were
first made on plasmid pCA2 by site-directed mutagenesis, and then the
petCA operon containing the desired mutation was excised from pCA2 by
PacI and XbaI digestion and cloned into the PacI
and XbaI sites of pCA1 to generate the pCA1 mutant.
To delete the petC1 gene in the petCA operon,
PmlI and SmaI unique sites were introduced at the 5'-
and 3'-ends of petC1 by site-directed mutagenesis with CT ATA
CAA AAG GCT CTG ATT ATG CAC GTG ACT CAA TTA TCA GGT TCC T and C CGT
ACT GAC GAA GCT CCC GGG TGG GCC TAA AAA CCC CAT ACG and their
complementary primers. The resulting pCA2 mutant was digested with
PmlI and SmaI to remove petC1 and religated to
generate plasmid pCA2-
petC1. The
petCA-
petC1 operon was then excised from
pCA2-
petC1 and cloned into pCA1 to generate
pCA1-
petC1. Mutations in the ISP hinge region were made by
site-directed mutagenesis with the following primers (complementary primers
not shown): P44A/P45A, CCC GTT ATT AAA TAT TTT ATT GCT GCT TCT AGT
GGT GGC; S46G/S47G, GTT ATT AAA TAT TTT ATT CCT CCT GGT GGT GGT GGC GCT GGT
GG; 4Pro
[PDB]
, CCT CCT TCT AGT GGT CCC CCT CCT CCT GGC GTG ATT GC; 6Pro,
CCC GTT ATT AAA TAT TTTT ATT CCT CCT CCT CCT GGT CCC CCT CCT
CCT GGC;
A50, CCT CCT TCT AGT GGT GGC_GGT GGT GGC GTG ATT GC;
S4647, GTT ATT AAA TAT TTT ATT CCT CCT__GGT GGC GCT GGT GGT GG;
G5152, CCT CCT TCT AGT GGT GGC GCT__GGC GTG ATT GCT AAA GAT GCC;
+2G-
2G, CCC GTT ATT AAA TAT TTT ATT CCT CCT GGC GGC TCT AGT
GGT GGC GCT__GGC GTG; and +4Gly, CCT TCT AGT GGT GGC GCT GGT GGC
GGC GGT GGT GGC GGC GTG ATT GC.
Transformation of Synechococcus Cells and Screening of Complete
SegregantsTransformation of Synechococcus PCC 7002 with
plasmid was performed as described
(30,
31), except that, after
incubation of the plasmid with cells at 37 °C under light for 90 min, the
plasmid/cell mixture was applied to medium A plates supplemented with 100
µg/ml spectinomycin. After 710 days of incubation,
spectinomycin-resistant transformants appeared and were streaked on
spectinomycin plates for further segregation. The primary screening for the
complete segregants with the desired mutation in the petC1 gene of
the Synechococcus genome was performed by PCR and subsequent
restriction analysis of the PCR product (see
Fig. 3A).
Synechococcus genomic DNA for PCR was extracted as described
(30,
35). A 1-kb fragment
containing petC1 and the 5'-part of petA was amplified
with a forward primer (GGG CAA ACT AAG GCT C) and a reverse primer (AAA CTT
GAT CGG GTA AAA CGG). The PCR product was digested with HindIII and
then analyzed by agarose gel electrophoresis. Complete digestion of the PCR
product with HindIII indicated complete segregation and absence of
the wild-type allele in the Synechococcus genome. The presence of the
desired mutations in the petC1 gene of the Synechococcus
genome and complete segregation were confirmed by sequencing of the PCR
products that could be completely digested with HindIII.

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FIG. 3. A, strategy for the introduction of the petC1 mutant into
the Synechococcus sp. PCC 7002 genome and the detection of its
segregation from the wild-type (WT) allele. After transformation, the
plasmid pCA1, containing the entire petCA operon with a new
HindIII site (silent mutation) at the 3'-end of petC1,
an upstream aadA cassette, and a 1.5-kb 5'-flanking sequence,
confers spectinomycin resistance on the host upon integration of the
aadA cassette and its closely linked mutated petC1 gene into
the Synechococcus genome. The primary screening for complete
segregants with the desired mutation in the petC1 locus of the
Synechococcus genome was performed by PCR and subsequent
HindIII restriction analysis of the PCR product. B, PCR and
HindIII restriction analysis of the genomic DNA from the wild type
(lane 1) and mutants P44A/P45A (lane 2), S46G/S47G (lane
3), 4Pro
[PDB]
(lane 4), A50 (lane 5),
S4647 (lane 6), G5152 (lane 7),
+2G- 2G (lane 8), +4Gly (lane 9), and
petC1 (lane 10). A 1-kb fragment containing
petC1 and the 5'-part of petA was amplified from the
Synechococcus genomic DNA of the transformants by PCR with a forward
primer (GGG CAA ACT AAG GCT C) and a reverse primer (AAA CTT GAT CGG GTA AAA
CGG). The PCR product was digested with restriction enzyme HindIII
and then analyzed by agarose gel electrophoresis.
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Oxygen EvolutionOxygen evolution rates were measured at 39
°C with a Clark-type oxygen electrode and a saturating actinic light
intensity of 20002500 microeinsteins/m2/s. Cells harvested
in late log phase were suspended at a chlorophyll concentration of 10 µg/ml
in 5 mM HEPES (pH 7.5), 10 mM NaCl, and 10 mM
NaHCO3.
Flash Kinetic SpectroscopyThe electron transfer activity of
the cytochrome b6 f complex in vivo was
measured in terms of the rate of flash-induced re-reduction of cytochromes
f and c6 in their
-band absorption region
(
A556-540 nm) using a
spectrometer as described
(36). Synechococcus
cells were harvested in late log phase and kept in darkness. Prior to
measurement, cells were suspended at 5 µM chlorophyll in
reaction medium consisting of 5 mM HEPES (pH 7.5), 10 mM
NaCl, and 10 mM NaHCO3. 10 µM
3-(3,4-dichlorophenyl)-1,1-dimethylurea, 1 mM NH2OH, and
10 µM carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone were added to dissipate
flash-induced absorbance changes from photosystem II and to disrupt the
transmembrane electrochemical potential. To eliminate variation in the redox
state of the plastoquinone pool in the dark, 1.0 mM KCN was
incubated at room temperature (1 min) to block respiration.
Homology Modeling of the Cytochrome b6 f
ComplexFrom the sequence similarity between the cytochrome
b in the bc1 complex and cytochrome
b6 /subunit IV and the positions of the invariant residues
(5,
37), it has been concluded
that cytochrome b6 and subunit IV should have the same
general structure as the N- and C-terminal domains of the mitochondrial
cytochrome b (5,
37). Also, from the high
degree of sequence conservation observed around the Qp site that
contacts the ISP and the nearly identical primary and tertiary structures of
the cluster-binding ISP small subdomain in chloroplasts and mitochondria
(9,
37), the ISP in the
stigmatellin-bound b6 f complex should be in a
similar position and orientation as observed in the stigmatellin-bound
bc1 complex. Therefore, three of the four large subunits
in the b6 f complex can be modeled within the
complex, whereas the position of the unique cytochrome f is not
known. A model of the b6 f complex in which the
ISP is located at the Qp site and cytochrome f is excluded
was built by homology modeling with SWISS MODEL and SWISS-pdb viewer
(38). The crystal structure of
the yeast bc1 complex with stigmatellin (Protein Data Bank
code 1EZV
[PDB]
) (15) was used as a
template for the homology modeling. The structure of the
Synechococcus ISP soluble domain was modeled from the crystal
structure of the spinach ISP soluble domain (Protein Data Bank code 1RFS
[PDB]
)
(9) and superimposed on the
complex based on the structural similarity of the cluster-binding small
subdomains between the mitochondrial and chloroplast ISPs
(9).
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RESULTS
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The petC1 Gene Encodes Functional ISPThe ISP of the
b6 f complex is encoded by the petC gene
in higher plants and cyanobacteria. Originally, only one petC gene
(petC1), which is located in the petCA operon, was reported
for Synechococcus sp. PCC 7002
(33). However, a BLAST search
in the recently deposited genome (NCBI accession number NC_003488) indicated
that, as in Synechocystis sp. PCC 6803
(39), there are two additional
petC genes (petC2 and petC3) in the
Synechococcus genome. In Synechocystis, the petC
gene located in the petCA operon was shown to be the dominant species
in membranes and in isolated b6 f complex by
immunoblotting (39). To study
the function of PetC1 and its hinge region, a
petC1 mutant
with the entire petC1 gene deleted and nine hinge region mutants with
changes in the composition, flexibility, and length of the hinge region were
constructed (Table II).
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TABLE II Cell growth and electron transfer activity of the wild type and hinge
region mutants of Synechococcus sp. PCC 7002
Data reported with S.D. were obtained from three or more trials; data
without S.D. were obtained from a single experiment. WT, wild type; ND, not
done.
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The cytochrome b6 f complex is required for
both respiration and photosynthesis in cyanobacteria; therefore, only
non-lethal mutants can be propagated and segregated from the wild-type allele,
and deleterious mutations are detected as non-segregants. All hinge region
mutants reported in this study (listed in
Table II) were segregated
completely from the wild type. The PCR products of their genomic
petC1 genes could all be digested completely with HindIII
(Fig. 3B, lanes
29) (data for mutant 6Pro not shown). All these mutants grew well
under photosynthetic conditions with a growth rate (doubling time of
4.56.2 h) similar to that of the wild type (doubling time of 5.4 h)
(Table II). Analysis of the
content of the b6 f complex in membranes by
Western blotting and heme staining indicated that the assembly and stability
of the b6 f complex were not significantly
affected in these mutants (data not shown). On the contrary, no segregant
could be obtained for the
petC1 mutant. Thus, both wild-type
and
petC1 alleles were present in the PCR products of
non-segregants (Fig.
3B, lane 10), implying an essential role of the
petC1 gene product for Synechococcus sp. PCC 7002. In
addition, certain mutations (e.g. deletion of Gly51 and
Gly52) in the PetC1 hinge region caused a significant decrease in
the electron transfer activity of the b6 f
complex and a loss of its sensitivity to DBMIB (discussed below), suggesting a
dominant role of PetC1 in the function of the b6
f complex. Therefore, it was concluded that petC1 codes for
the essential ISP in Synechococcus sp. PCC 7002, whereas
petC2 and petC3 are silent in the function of the
b6 f complex. This makes it practical to analyze
the structure and function of the ISP by mutagenesis of petC1 without
knockout of petC2 and petC3 in Synechococcus sp.
PCC 7002.
Flash-induced Cytochrome f/c6
KineticsIn Synechococcus sp. PCC 7002, cytochrome
c6, instead of plastocyanin, whose gene cannot be found in
the genome (NCBI accession number NC_003488), mediates electron transfer from
the cytochrome b6 f complex to photosystem I. The
electron transfer activity of the cytochrome b6 f
complex in vivo can be measured in terms of the rate of flash-induced
re-reduction of cytochromes f and c6 in their
-band absorption region by a flash spectrometer with intact
Synechococcus cells
(30,
40). A light-dark difference
spectrum of flash-induced absorbance changes in the region at 540570 nm
clearly shows the absorbance changes to consist of contributions from both
cytochromes f (
-band maximum, 556 nm) and
c6 (
-band maximum, 553 nm) (data not shown). The
observations in this study that (i) deletion of 2 residues at two different
positions in the hinge region produced similar effects on the kinetics of
cytochromes f and c6 and (ii) the retardation of
the cytochrome f/c6 reduction and the loss of its
sensitivity to inhibitors in mutant
G5152 could be fully
reverted by reinsertion of 2 Gly residues at a second site between
Pro45 and Ser46 in a "restoration mutant,"
+2G-
2G (Fig. 4 and
Table II; discussed below),
show that the flash-induced absorbance change in this region is fully
attributed to the electron transfer activity of the b6
f complex.
Consequences of Increased Flexibility and Length of the Hinge
RegionIt has been shown in Rhodobacter capsulatus that
introduction of excess flexibility by substitution with 6 Gly residues
(19) or of extra length by
insertion of 1 or more residues
(17,
24,
26) in the hinge region is
deleterious to the function of the bc1 complex
(Table III, parts A and B).
With 47 glycine residues in the sequence, the ISP hinge region in the
b6 f complex is predicted to be structure-less
and more flexible than that in the bc1 complex
(Table I). Further increases in
flexibility or length caused by changing Pro44 and
Pro45, which are conserved at the N-terminal end of the hinge
region but absent in the bc1 sequences, to 2 Ala residues
in mutant P44A/P45A or Ser46 and Ser47 to 2 Gly residues
in mutant S46G/S47G or by insertion of 4 extra Gly residues in mutant +4Gly
(resulting in a total of 9 Gly residues in the "hinge" region) had
no effect on the activity of the complex. These mutants showed an electron
transfer activity (t1/2 of cytochrome
f/c6 reduction = 3.15.2 ms) similar to
that of the wild type (t1/2 of cytochrome
f/c6 reduction = 4 ms)
(Fig. 4 and
Table II). Therefore, it is
concluded that, in contrast to the bc1 complex, extreme
flexibility and extra length of the hinge region are well accommodated in the
b6 f complex
(Table III, parts A and B).
Effect of Decreased FlexibilityIn the bacterial
bc1 complex, substitution of 26 Pro residues in the
hinge region greatly inhibited the activity
(Table III, part A), which was
considered key evidence in support of a functional requirement for mobility of
the ISP soluble domain (19,
23,
41). Because flexibility of
the ISP hinge region associated with the polyglycine sequence motif appears to
be a conserved characteristic of the b6 f complex
hinge region (Table I), it was
expected that a high degree of flexibility would be critical for the ISP
movement. Surprisingly, decreasing the flexibility of the hinge region by
substitution of 4 Pro residues for residues 4952 (GAGG, mutant 4Pro
[PDB]
)
failed to inhibit the electron transfer rate of the complex
(t1/2 of cytochrome f/c6
reduction = 3.7 ms) (Fig. 4 and
Table II). A conformationally
inflexible mutant was made by substitution of 2 additional proline residues
for Ser46 and Ser47 in mutant 6Pro, resulting in a
linker region with 8 Pro residues (including the native Pro44 and
Pro45 residues) (Table
II). There was a decrease in activity associated with this mutant,
but the 2.4-fold decrease in the electron transfer rate
(t1/2 of cytochrome f/c6
reduction = 9.6 ms) (Fig. 4 and
Table II) was small considering
the severity of the mutation and the effect of such mutations on the
bc1 complex (Table
III, part A). Apparently, a high degree of rigidity in the hinge
region is well tolerated in the b6 f complex. The
relatively small perturbation caused by a Gly6 -to-Ala6
substitution, generated in other studies, has no effect on the function of the
b6 f complex in the green alga Chlamydomonas
reinhardtii (42).
Consequences of Shortening the Hinge RegionIn the bacterial
and yeast cytochrome bc1 complexes, deletion mutations in
the ISP hinge region resulted in a 40100% decrease in activity and
often defects in assembly or stability
(Table III, part C)
(19,
23,
24,
26). In the
b6 f complex, the 1-residue deletion mutant
A50 displayed a phenotype similar to that of the wild type
(t1/2 of cytochrome f/c6
reduction = 4.4 ms) (Fig. 4 and
Table II), whereas the
bc1 complex was sensitive to a 1-residue deletion
(Table III, part B). The rate
of cytochrome f/c6 reduction was decreased by
factors of 2.5 and 4 compared with the wild type in the 2-residue deletion
mutants
S4647 (t1/2 = 10 ms) and
G5152 (t1/2 = 15 ms)
(Fig. 4 and
Table II), respectively.
Although the decreased activity of the hinge region mutant truncated by 2
residues implies that the minimum length for full activity is 7 residues, the
2-residue truncation mutation is not severe, as mutants could still grow and
evolve oxygen at the wild-type rate (Fig.
5 and Table II). This is presumably because the rate-limiting step of electron transport under
conditions of coupled phosphorylation for cell growth and O2
evolution is slower than that under the uncoupled conditions of flash-induced
turnover. Because deletion of 2 residues at two different positions in mutants
S4647 and
G5152 produced similar phenotypes, the
observed effect of 2-residue deletions is attributed to truncation in length
instead of a positional effect. This was confirmed by the restoration mutant
+2G-
2G for mutant
G5152, in which the reinsertion of 2
Gly residues at a second site between Pro45 and Ser46
could fully revert the loss of function in
G5152
(Fig. 4 and
Table II). The inference that 7
residues is the minimum length of the hinge region that retains full function
is confirmed by studies with Qp site inhibitors.
Sensitivity of Hinge Region Mutants to Qp
Site Inhibitors Electron transfer activity was measured after
flash excitation in the presence of the Qp site inhibitors DBMIB
and stigmatellin. The wild type and mutants P44A/P45A, S46G/S47G, 4Pro
[PDB]
,
A50, and +2G-
2G were sensitive to DBMIB to a similar extent
(Table II). 2.5 and 20
µM DBMIB inhibited cytochrome f/c6
re-reduction, slowing the reduction half-times for these strains from
35 ms to 130260 and 4101080 ms, respectively. Mutant 6Pro
was slightly less sensitive to DBMIB, with 2.5 µM DBMIB slowing
the reduction half-time from 9.6 to 80 ms
(Table II). However, the
2-residue truncation mutants
S4647 and
G5152 were
insensitive to DBMIB (Table
II). 20 µM DBMIB inhibited cytochrome
f/c6 re-reduction only slightly, slowing the
half-times in mutants
S4647 and
G5152 from 10 to
18 ms and from 15 to 18 ms, respectively. The loss of inhibitor sensitivity in
the latter mutant was reverted in the restoration mutant +2G-
2G
(Table II). A similar result
was obtained for O2 evolution, where 2.5 µM DBMIB
caused >95% inhibition in the wild type, but only
30% in mutants
S4647 and
G5152
(Fig. 5). Similarly, the
cytochrome b6 f complexes in mutants
S4647 and
G5152 were insensitive to stigmatellin.
10 µM stigmatellin caused an
10-fold decrease in the
cytochrome f/c6 reduction rate in the wild type,
but only an
1.5-fold decrease in these two mutants
(Fig. 6). The loss of
sensitivity to Qp site inhibitors in the 2-residue truncation
mutant is consistent with structure data showing that the cluster-binding
subdomain of the ISP is required to form the inhibitor-binding site, the
Qp site (Fig. 1). It
is inferred that a hinge region shorter than 7 residues results in
insufficient contact between the ISP cluster-binding region, the membrane
surface, and an altered Qp site.
In contrast to the loss of inhibitor sensitivity in the 2-residue deletion
mutants, the b6 f complex became more sensitive
to Qp site inhibitors in mutant +4Gly. 2.5 µM DBMIB
caused an
70-fold decrease in the cytochrome
f/c6 reduction rate in the wild type and an
almost 400-fold decrease in mutant +4Gly
(Table II). A similar result
was also obtained in flash experiments with stigmatellin
(Fig. 6). Thus, the pronounced
elongation allows a different fit of the ISP to the Qp-binding
niche, resulting in an increase in affinity for quinol analog inhibitors, but
changes in distance from the Qp site quinol to the [2Fe-2S] cluster
and its ligands are small enough that the measured electron transfer rate is
not affected.
 |
DISCUSSION
|
---|
Insensitivity of the b6 f Complex to Hinge
Region Structure Changes: Implications for FunctionThe
bc1 and b6 f complexes
display a very different sensitivity to changes in flexibility and increases
in length of their hinge regions (Table
III, parts A and B). Decreasing the flexibility of the hinge
region by substitution of 26 Pro residues or introduction of a
disulfide bond in this region caused a large decrease in the activity of the
bc1 complex, consistent with the concept that these
changes hamper the mobility and movement of the ISP soluble domain
(19,
22,
23). However, the deleterious
effects of an increase in flexibility of the hinge region by substitution of 6
Gly residues (19) and of
increases in length by insertion of 13 Ala residues
(17,
24,
26) are not readily explained
by the x-ray structures or the concept of preservation of ISP mobility.
Indeed, although x-ray structure data led to the concept of the mobile ISP as
an essential component in the high potential electron transport pathway for
quinol oxidation (7,
12,
13,
14,
15,
16), these structure data do
not at present elucidate the mechanism that drives and controls the ISP
motion. The hinge region mutagenesis data in the bc1
complex imply that there is an optimum structure of the hinge region for
mobility and function. In the b6 f complex, the
8-residue ISP hinge region consists mostly of Gly residues and is presumably
more flexible than that in the bc1 complex
(Table I). The present study
indicates that an optimum flexibility and length of the hinge region are not
required for the function of the b6 f complex. It
can be mutated to be conformationally more flexible, extremely rigid (up to
substitution of 6 Pro residues), or 4 residues longer without significant
effect on function (Tables II
and III). The large difference
between the b6 f and bc1
complexes in their responses to structure changes in the ISP hinge region
implies a significant difference in the mechanism and/or amplitude of the ISP
movement in the two complexes.
Physical Properties of ISP MotionIn the crystal structures
of the bc1 complex, the hinge region is uncoiled and
extended when the ISP [2Fe-2S] cluster is in the quinol-proximal position
(Qp or "b" position), whereas it curls up in a
helical conformation when it is proximal to cytochrome c1
(Fig. 1)
(12,
14,
15). It is not clear whether
such a secondary structure change in the hinge region is a driving force for,
or solely a result of, the ISP domain movement in the bc1
complex. Considering the large differences between the bc1
and b6 f complexes in the structures of their
hinge regions and their responses to hinge region mutations, it is very likely
that formation of the helix in the bc1 ISP hinge region is
needed to drive the reduced ISP [2Fe-2S] cluster away from the Qp
site toward the "c1" position, whereas such a
driving device is apparently absent in the b6 f
complex. This hypothesis would explain the observation in the
bc1 structures that the ISP is in an intermediate or
c1 position in the absence of stigmatellin
(12,
14) because of favorable helix
formation in the hinge region. It also explains the high sensitivity of the
bc1 complex to changes in flexibility and increases in
length of the hinge region. Changes in flexibility by Pro or Gly substitutions
or introduction of a disulfide bond in the hinge region
(19,
22,
23) would interrupt the
formation of
-helical structure in the hinge region and thus impair ISP
movement. An increase in hinge length
(17,
24,
26) would decrease the tension
in the hinge region, making it unable to tug the ISP away from Qp
site, as was suggested previously
(16). In the case of the hinge
region of the b6 f complex in wild-type
Synechococcus sp. PCC 7002, it should be essentially structure-less
because of the presence of 5 Gly residues and 2 adjacent Pro residues
(Table I), implying that
secondary structure change or tension in the linker region is not required for
ISP domain movement in the b6 f complex.
Therefore, excess flexibility and length are not inhibitory. However, the
finding that the large decreases in hinge flexibility by substitution of
46 Pro residues have at most a small effect on the function of the
b6 f complex is surprising. It suggests that
hinge flexibility is less important than in the bc1
complex.
Why is a coil-helix transition mechanism not required, and a high degree of
hinge rigidity allowed in the b6 f complex? We
propose two alternative explanations. (i) A structural barrier for ISP
movement that is present in the bc1 complex is absent or
smaller in the b6 f complex. The
"ef loop" in the bc1 complex connects
the most separated transmembrane helices, E and F, of cytochrome b on
the p side of the membrane and holds the ISP soluble domain and
cytochrome c1 in the correct orientation for ISP movement
and electron transfer. Substitution of Phe for Leu286 (located
around site b in Fig.
2A) in the ef loop can partially overcome the
defect in ISP movement in mutant +1Ala of R. capsulatus, indicating
that this surface loop represents the major physical barrier to ISP domain
movement from the Qp site to the c1 position
(21). Leu286
(Leu262 in the bovine complex) is highly conserved in the
bc1 complex, but is substituted with a highly conserved
Phe (Phe69 in subunit IV in Synechococcus PCC 7002) in the
b6 f complex. Thus, the physical barrier in the
ef loop of the bc1 complex may be absent in the
b6 f complex. In the absence of the barrier, the
coil-helix transition of the hinge region may not be required to pull the ISP
from the Qp site to the c1 positions.
(ii) The studies on changes in orientation of the [2Fe-2S] cluster in the
presence and absence of DBMIB
(27) show that the ISP
movement is sufficient to reorient the principle g value transitions of the
[2Fe-2S] cluster. However, the amplitude of the ISP cluster motion may be
significantly smaller than the 60 °-16 Å rotation-translation
inferred for the bc1 complex. A smaller amplitude would
allow decreased flexibility and smaller conformational constraints in the
hinge region. An important structure difference between the
b6 f and bc1 complexes is
that the electron acceptors of the ISP, cytochromes f and
c1, respectively, are completely different in sequence and
structure (10,
11) and heme orientation
(43,
44,
45,
46). A close orientation of
the cytochrome f heme to the ISP [2Fe-2S] cluster would eliminate the
necessity of large-scale ISP movement; on the other hand, a steric constraint
from cytochrome f on the free space for ISP movement would prevent
large-scale ISP movement, although a significant displacement of the ISP
[2Fe-2S] cluster from the quinol of the Qp site is still required
for a bifurcated electron transport chain in both cases
(7).
Minimum Length of the Hinge Region Required for Optimum Binding of ISP
at the Qp SiteThe loss of sensitivity
of the b6 f complex to the Qp site
inhibitors in the 2-residue truncation mutants is striking. Previously, only a
small (50%) increase in the I50 of stigmatellin was
reported for a 1-residue truncation mutant of the yeast mitochondrial
bc1 complex
(26), and no information on
changes in inhibitor sensitivity was provided from studies on photosynthetic
bacteria (Table III)
(17,
18,
19,
21,
22,
23). However, alterations in
EPR spectra of the ISP were observed in some bc1 hinge
region truncation mutants of the bacteria
(19,
23). These results indicate
that truncation of the hinge region has a direct impact on the structure of
the Qp site in both the b6 f and
bc1 complexes. The observed decrease in
b6 f/bc1 activity in these
truncation mutants can be readily explained by defects in the Qp
site that also result in a decrease in the Km of
quinol, as was observed in the yeast mitochondrial bc1
complex (26).
Examination of the interaction between the ISP soluble domain and the
p side of cytochrome b in the presence of stigmatellin
indicates that a minimum hinge length is sterically required for the proper
docking of the [2Fe-2S] cluster region at the Qp site. In the
structure, the ISP soluble domain resembles a lever, with the [2Fe-2S] cluster
region located at one end and the hinge region linked to the other end
(Fig. 2A). In the
middle, the ISP
23-strand region (site a in
Fig. 2) is positioned close to
the ef loop of cytochrome b (site b in
Fig. 2) to form a
"fulcrum region" (Fig.
2). Because of the steric repulsion between the ISP and cytochrome
b in the fulcrum region, the moving and binding of the [2Fe-2S]
cluster region to the membrane (Qp site) would lead to displacement
of the other end of the ISP soluble domain and elongation of the hinge region.
Vice versa, truncation of the hinge region would hinder the movement and
binding of the [2Fe-2S] cluster region to the Qp site. This fulcrum
region in the bc1 complex may also work as the
"structure barrier region" of the ISP domain movement proposed in
Ref. 21 (see discussion
above). The structure and orientation of the
23-strand
region are similar in the bc1, b6
f, and archaeal Rieske ISPs
(9,
47). A similar pattern of
interaction between the ISP soluble domain and the p side of
cytochrome b6 /subunit IV was observed in the modeled
structure of the b6 f complex
(Fig. 2B). Thus, it is
inferred that a minimum length of 7 residues in the ISP hinge region is
required for optimum binding of the ISP at the Qp site in the
b6 f complex. The decrease in activity and the
loss of Qp site inhibitor sensitivity in the 2-residue truncation
mutants may be explained by the requirement for H-bonding of quinol and
stigmatellin to the His129 (equivalent to His161 in
bovine) ligand of the [2Fe-2S] cluster for efficient electron transfer from
quinol to ISP and inhibitor binding
(12,
15,
48). A truncation of the hinge
region would not allow close approach of the [2Fe-2S] cluster at the
Qp site and would thus impair the formation of this H-bond.
 |
FOOTNOTES
|
---|
* This work was supported by National Institutes of Health Grant GM-38323.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 765-494-4956; Fax:
765-496-1189; E-mail:
wac{at}bilbo.bio.purdue.edu.
1 The abbreviations used are: ISP, iron-sulfur protein; DBMIB,
2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. 
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to G. M. Soriano and H. Zhang for helpful advice and
discussions and T. Kallas and W. R. Widger for kind gifts of the
Synechococcus sp. PCC 7002 wild-type strain and plasmid pUHWRW1,
respectively.
 |
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