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 {ddagger}

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.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 4–6 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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}-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 {alpha}-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.

 

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 4–7 Gly, 0–3 Ser, 0–1 Thr, and 0–2 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 {beta}2–3-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).

 


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth of Cultures—Synechococcus 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 Construction—A 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-{Delta}petC1. The petCA-{Delta}petC1 operon was then excised from pCA2-{Delta}petC1 and cloned into pCA1 to generate pCA1-{Delta}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; {Delta}A50, CCT CCT TCT AGT GGT GGC_GGT GGT GGC GTG ATT GC; {Delta}S46–47, GTT ATT AAA TAT TTT ATT CCT CCT__GGT GGC GCT GGT GGT GG; {Delta}G51–52, CCT CCT TCT AGT GGT GGC GCT__GGC GTG ATT GCT AAA GAT GCC; +2G-{Delta}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 Segregants—Transformation 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 7–10 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), {Delta}A50 (lane 5), {Delta}S46–47 (lane 6), {Delta}G51–52 (lane 7), +2G-{Delta}2G (lane 8), +4Gly (lane 9), and {Delta}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.

 

Oxygen Evolution—Oxygen evolution rates were measured at 39 °C with a Clark-type oxygen electrode and a saturating actinic light intensity of 2000–2500 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 Spectroscopy—The 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 {alpha}-band absorption region ({Delta}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 Complex—From 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The petC1 Gene Encodes Functional ISP—The 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 {Delta}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.

 

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 2–9) (data for mutant 6Pro not shown). All these mutants grew well under photosynthetic conditions with a growth rate (doubling time of 4.5–6.2 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 {Delta}petC1 mutant. Thus, both wild-type and {Delta}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 Kinetics—In 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 {alpha}-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 540–570 nm clearly shows the absorbance changes to consist of contributions from both cytochromes f ({alpha}-band maximum, 556 nm) and c6 ({alpha}-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 {Delta}G51–52 could be fully reverted by reinsertion of 2 Gly residues at a second site between Pro45 and Ser46 in a "restoration mutant," +2G-{Delta}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.



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FIG. 4.
Kinetics of flash-induced cytochrome f/c6 redox changes in vivo in the wild type and ISP hinge region mutants in the absence of inhibitors. Each trace is an average of 36 flash-induced absorbance changes with 2 s of darkness between runs. {Delta}A(cytochrome f/c6) = {Delta}A(556 nm) - {Delta}A(540 nm). Cells were suspended at 5 µM chlorophyll in 5 mM HEPES (pH 7.5), 10 mM NaCl, and 10 mM NaHCO3. 10 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, 1 mM NHOH, 10 µM carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, and 1.0 mM KCN were added prior to measurements. Onset of flash is indicated by the arrows. The strain used for each measurement is shown at the top of each panel. WT, wild type.

 

Consequences of Increased Flexibility and Length of the Hinge Region—It 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 4–7 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 = 3.1–5.2 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).


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TABLE III
Comparison of effects of ISP hinge region mutations on cytochrome bc1 and b6f complexes

 

Effect of Decreased Flexibility—In the bacterial bc1 complex, substitution of 2–6 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 49–52 (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 Region—In the bacterial and yeast cytochrome bc1 complexes, deletion mutations in the ISP hinge region resulted in a 40–100% 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 {Delta}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 {Delta}S46–47 (t1/2 = 10 ms) and {Delta}G51–52 (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 {Delta}S46–47 and {Delta}G51–52 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-{Delta}2G for mutant {Delta}G51–52, in which the reinsertion of 2 Gly residues at a second site between Pro45 and Ser46 could fully revert the loss of function in {Delta}G51–52 (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.



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FIG. 5.
Oxygen evolution rates of the wild-type and mutant {Delta}S46–47 and {Delta}G51–52 cells in the absence and presence of 2.5 µM DBMIB. Oxygen evolution rates were measured at 39 °C as described under "Materials and Methods." Chl, chlorophyll; WT, wild type.

 

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] , {Delta}A50, and +2G-{Delta}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 3–5 ms to 130–260 and 410–1080 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 {Delta}S46–47 and {Delta}G51–52 were insensitive to DBMIB (Table II). 20 µM DBMIB inhibited cytochrome f/c6 re-reduction only slightly, slowing the half-times in mutants {Delta}S46–47 and {Delta}G51–52 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-{Delta}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 {Delta}S46–47 and {Delta}G51–52 (Fig. 5). Similarly, the cytochrome b6 f complexes in mutants {Delta}S46–47 and {Delta}G51–52 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.



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FIG. 6.
Effect of stigmatellin on the electron transfer activity of the b6 f complex in the wild-type and mutants {Delta}S46–47, {Delta}G51–52, and +4Gly. The relative electron transfer activity of the b6 f complex as a function of stigmatellin concentration is expressed as a ratio of the flash-induced re-reduction rates of cytochrome f/c6 measured in the presence and absence of stigmatellin. Flash kinetic measurements were performed as described in the legend to Fig. 4. WT, wild type.

 

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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Insensitivity of the b6 f Complex to Hinge Region Structure Changes: Implications for Function—The 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 2–6 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 1–3 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 Motion—In 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 {alpha}-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 4–6 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 Site—The 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 {beta}2–3-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 {beta}2–3-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. Back

{ddagger} 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. Back


    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.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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