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A R Crofts

Publications and source records attributed to A R Crofts.

At least 37 records · Page 2Linked to original sources
PubMed1998

Structure and function of the cytochrome bc1 complex of mitochondria and photosynthetic bacteria.

Progress has recently been made in the understanding of the function of the cytochrome bc1 complex and related proteins in the context of recent structural information. The structures support many features that were predicted from sequence analysis and biophysical studies, but contain some surprises. Most dramatically, it is apparent that the iron-sulfur protein can take up different positions in different crystals, suggesting a novel mechanism for electron transfer through domain movement. Evidence from studies of mutant strains, in which the function of the sites or the binding of inhibitors is perturbed, has provided clues about the mechanism.

Animals↗
PubMed1997

Reactions of isocytochrome c2 in the photosynthetic electron transfer chain of Rhodobacter sphaeroides.

Rhodobacter sphaeroides strains lacking cytochrome c2 (cyt c2), the normal electron donor to P870+ in light-oxidized reaction center (RC) complexes, are unable to grow photosynthetically. However, spd mutations that suppress the photosynthetic deficiency of cyt c2 mutants elevate levels of the cyt c2 isoform, isocyt c2. We monitored photosynthetic electron transfer in whole cells, in chromatophores, and with purified components to ascertain if and how isocyt c2 reduced light-oxidized RC complexes. These studies revealed that several fundamental aspects of photosynthetic electron transfer were similar in strains that use isocyt c2 and wild-type cells. For example, P870+ reduction accompanied cytochrome c oxidation. In addition, photosynthetic electron transfer was blocked by the well-known cyt bc1 complex inhibitors antimycin and myxothiazol. However, even at the increased isocyt c2 levels present in these strains (approximately 40% that of cyt c2 in wild-type cells), there was little, if any, of the rapid (< 5 microns) electron transfer to P870+ that is characteristic of cytochromes bound to RC complexes at the time of the light flash. Thus, it appears that isocyt c2 function limits the in vivo rate of P870+ reduction. Indeed, at low ionic strength in vitro, the apparent affinity of isocyt c2 for RC complexes (KD approximately 40 microM) is significantly lower than that of cyt c2 (KD approximately 1.0 microM). This reduced affinity does not appear to result from an altered mode of RC binding by isocyt c2 since electrostatic interactions make similar overall contributions to the binding of both cyt c2 and isocyt c2 to this membrane-bound redox partner. Thus, sequence, structural, or local conformational differences between cyt c2 and isocyt c2 significantly alter their apparent affinities for this physiologically relevant redox partner.

Cytochrome c Group↗
PubMed1996

Donor-side photoinhibition in photosystem II from Chlamydomonas reinhardtii upon mutation of tyrosine-Z in the D1 polypeptide to phenylalanine.

When tyrosine-Z of the D1-polypeptide of the photosystem II from Chlamydomonas reinhardtii was changed to phenylalanine, the rapid donor to P680+ was lost, and P680+ accumulated on illumination. The rapid donation from tyrosine-Z was replaced by a slow electron transfer from an endogenous donor. Spectrophotometric measurements showed that carotenoids and chlorophylls were bleached by the P680+ either directly or indirectly upon illumination. The carotenoid bleaching was inhibited in the presence of SOD or catalase, but the reaction did not require molecular oxygen as an electron acceptor. These observations led us to conclude that active oxygen radicals, possibly hydroxyl radicals, take part in the destruction of carotenoids in the Y161F mutant. Possible mechanisms for the destruction are discussed.

Algal Proteins↗
PubMed1994

Site-directed mutagenesis of arginine-114 and tryptophan-129 in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides: two highly conserved residues predicted to be near the cytoplasmic surface of putative transmembrane helices B and C.

The cytochrome b subunit of the ubiquinol:cytochrome c oxidoreductase (the bc1 complex) contains two heme prosthetic groups, cytochrome bL and cytochrome bH. In addition, this subunit also provides major elements of the quinol oxidation site (Qo) and a separate quinone reductase site (Qi), which are thought to be located on opposite sides of the membrane. Site-directed mutagenesis has been used to explore the role(s) of specific amino acid residues in this subunit from the photosynthetic bacterium Rhodobacter sphaeroides. Previous work identified five residues, Gly48 (Gly33), Ala52 (Gly37), His217 (His202), Lys251 (Lys228), and Asp252 (Asp229), as being either at or near the quinone reductase site (the residue numbers in parentheses designate the equivalent positions in the yeast mitochondrial enzyme). These residues are predicted to be near the cytoplasmic boundaries of transmembrane helices: helix A (G48, A52), helix D (H217), or helix E (K251, D252). In the current work, the importance of two additional highly conserved residues, which are also predicted to be near the cytoplasmic boundaries of transmembrane helices, is explored by site-directed mutagenesis. R114 (helix B) has been substituted with K, Q, and A, and W129 (helix C) has been changed to A and F. The results suggest that a positively charged residue at position 114 is important. The R114K mutation causes only subtle effects, which appear to be localized to cytochrome bH and the quinone reductase site. In contrast, R114Q is not assembled, and R114A, although partially assembled, is nonfunctional and appears to have a very low amount of cytochrome b associated with the complex. Both mutants at position 129 (W129A and W129F) are able to support the photosynthetic growth of the organism, but show abnormal characteristics. The defects associated with the W129A mutation appear to be primarily associated with the quinone reductase site and cytochrome bH, whereas the W129F mutation appears to result in more global defects that also perturb the cytochrome bL locus. The results are consistent with the placement of residues R114 and W129 near the cytoplasmic side of the membrane, but suggest that these residues are important for the assembly and overall stability of the complex.

Amino Acid Sequence↗
PubMed1994

A molecular mechanism for qE-quenching.

We discuss energy-dependent fluorescence lowering (qE-quenching), and suggest a model to explain the experimental data currently available. The main elements of the model are: (a) the qE-quenching reflects a mechanism associated with a component of the light-harvesting antenna rather than the reaction center of photosystem (PS) II--we suggest that it occurs through formation of an efficient quencher in one of the minor chlorophyll protein (CP) complexes; (b) the minor CPs have glutamate residues instead of glutamines at positions shown in light-harvesting complex II (LHCII) to be ligands to chlorophylls near the lumenal interface. We suggest that the quenching reflects a change in ligation of chlorophyll on protonation of these glutamate residues leading to formation of an exciton coupled dimer with a neighboring pigment, in which additional energy levels allow vibrational relaxation of the excited singlet. The model accounts for the dependence on low lumenal pH, the ligand residue changes between LHCII and the minor CPs, the preferential distribution of components of the xanthophyll cycle in the minor CPs, the inhibition of qE-quenching by DCCD, and the specific binding of DCCD to the minor CPs.

Amino Acid Sequence↗
PubMed1993

Site-directed mutations of conserved residues of the Rieske iron-sulfur subunit of the cytochrome bc1 complex of Rhodobacter sphaeroides blocking or impairing quinol oxidation.

Site-directed mutations of conserved residues in the domain binding the 2Fe-2S cluster of the Rieske subunit of the ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides have been constructed. The substitution of aspartate for glycine at position 133 in the Rb. sphaeroides sequence (mutant FG133D), which mimicked a mutation previously isolated and characterized in yeast by Gatti et al. [Gatti, D.L., Meinhardt, S.W., Ohnishi, T., & Tzagoloff, A. (1989) J. Mol. Biol. 205, 421-435], allowed more detailed studies of thermodynamic behavior and the kinetics of the ubiquinol:cytochrome c2 oxidoreductase on flash activation of the photosynthetic chain. The impaired catalysis in this mutant complex is localized to the quinol oxidizing site. The apparent second-order rate constant for reduction of cytochrome bH via the quinol oxidizing site is about 20-fold lower than that of the wild-type and correlates with its apparent activation barrier being increased relative to that of the wild-type. Substitutions for the cysteines and a histidine which are conserved in the putative 2Fe-2S binding domain of the Rieske subunit selectively knock out the 2Fe-2S cluster and quinol oxidizing activity, while leaving the cytochromes and other catalytic sites essentially intact. Reversion properties of these strains are consistent with the mutated residues being essential.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Characterization of mutations in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides that affect the quinone reductase site (Qc).

The cytochrome b subunit of the bc1 complex contains two heme components, cytochrome bL and cytochrome bH, and is the locus of both a quinol oxidizing site (Qo or Qz) and a quinone reducing site (Qi or Qc). The quinone reductase site has been previously characterized as the site of interaction for a set of inhibitors including antimycin A, diuron, funiculosin, and HQNO. In this paper, four highly conserved residues in the cytochrome b subunit of Rhodobacter sphaeroides (A52, H217, K251, and D252) were targeted for site-directed mutagenesis. These residues were chosen as being likely to be at or near the quinone reductase site, on the basis of known locations of missense mutations in the homologous yeast subunit that confer resistance to Qc-directed inhibitors. The site-directed mutants all exhibit a normal rate of reduction of cytochrome bH, suggesting a fully functional quinol oxidizing site. However, each of the mutants is impaired, to varying degrees, in the rate of reoxidation of cytochrome bH. Two mutants (H217A and D252A) are unable to grow photosynthetically, indicating a severe defect in the bc1 complex. In both cases, the cause of the defect is the lack of reoxidation of cytochrome bH by ubiquinone. This is the first report of mutations that selectively impair the rate of electron transfer from cytochrome bH to the Qc-site. This set of mutations will be useful not only for modeling the structure of the quinone reducing site but also in elucidating the catalytic mechanism of this portion of the Q-cycle.

Amino Acid Sequence↗
PubMed1993

Assembly of the Rieske iron-sulfur subunit of the cytochrome bc1 complex in the Escherichia coli and Rhodobacter sphaeroides membranes independent of the cytochrome b and c1 subunits.

The Rieske iron-sulfur subunit of the cytochrome bc1 complex from Rhodobacter sphaeroides has been expressed in Escherichia coli and also in a strain of Rb. sphaeroides lacking the other subunits of the bc1 complex. PCR products encoding the full-length subunit were introduced into expression vectors to produce the subunit alone or the subunit fused behind the mature portion of the E. coli maltose binding protein (MBP), but lacking the MBP signal sequence. These proteins are both located in the cytoplasmic membrane. The unfused Rieske subunit assembles a Rieske-like iron-sulfur cluster, but with EPR characteristics which differ from the normal rhombic signal observed in the cytochrome bc1 complex. The overproduced MBP fusion protein, on the other hand, does not contain an EPR-detectable iron-sulfur cluster. Subfragments of the Rieske subunit lacking the amino-terminal hydrophobic anchor also lack the iron-sulfur cluster were expressed in E. coli. When expressed in Rb. sphaeroides in the absence of the cytochrome b and c1 subunits, the fully metalated Rieske subunit with the diagnostic gy = 1.90 EPR signal is observed in the cytoplasmic membrane. The fact that the Rieske subunit has an assembled iron-sulfur cluster and is bound to either the E. coli or the Rb. sphaeroides membrane in the absence of the other subunits of the bc1 complex demonstrates a mode of membrane attachment independent of the other components of the complex. These data are consistent with models in which the Rieske subunit is bound to the membrane via a single membrane-spanning helix located near the amino terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP-Binding Cassette Transporters↗
PubMed1993

The bc1 complexes of Rhodobacter sphaeroides and Rhodobacter capsulatus.

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochrome c oxidoreductase (bc1 complex). In both Rhodobacter sphaeroides and Rhodobacter capsulatus, the bc1 complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because the bc1 complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochrome b subunit, in the Rieske iron-sulfur subunit, and in the cytochrome c1 subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Qo) site or the quinol reductase (Qi) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.

Amino Acid Sequence↗
PubMed1992

Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides.

The cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol-oxidizing site (Qo or Qz) and a quinone-reducing site (Qi or Qc). Sequence alignments of this subunit from over 20 eukaryotic and prokaryotic species have revealed a remarkable degree of conservation, including approximately 20 totally conserved residues. In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of 5 of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments. The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium. The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties and on the kinetics of specific electron transfer reactions. The results show that none of the 5 residues is absolutely essential. Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth. The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a dysfunctional quinone reductase (Qc) site; an alanine is tolerated at this position. Possibly, a small residue is important here for heme packing. Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands. Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes. Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes. Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the cytochrome b chain containing the two hemes. Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the 2 histidines which provide ligands to the hemes. No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b cytochromes and their electrochemical properties were noted.

Amino Acid Sequence↗
PubMed1992

Pressure and low temperature effects on the fluorescence emission spectra and lifetimes of the photosynthetic components of cyanobacteria.

The effects of hydrostatic pressure on the excited state reactions of the photosynthetic system of cyanobacteria were studied with the use of stationary and dynamic fluorescence spectroscopy. When the cells were excited with blue light (442 nm), hydrostatic pressure promoted a large increase in the fluorescence emission of the phycobilisomes (PBS). When PBS were excited at 565 nm, the shoulder originating from photosystem II (PSII) emission (F685) disappeared under 2.4 kbar compression, suggesting suppression of the energy transfer from PBS to PSII. At atmospheric pressure, the excited state decay was complex due to energy transfer processes, and the best fit to the data consisted of a broad Lorentzian distribution of short lifetimes. At 2.4 kbar, the decay data changed to a narrower distribution of longer lifetimes, confirming the pressure-induced suppression of the energy transfer between the PBS and PSII. When the cells were excited with blue light, the decay at atmospheric pressure was even more complex and the best fit to the data consisted of a two-component Lorentzian distribution of short lifetimes. Under compression, the broad distribution of lifetimes spanning the region 100-1,000 ps disappeared and gave rise to the appearance of a narrow distribution characteristic of the PBS centered at 1.2 ns. The emission of photosystem I underwent 2.2-fold increase at 2.4 kbar and room temperature. A decrease in temperature from 20 to -10 degrees C at 2.4 kbar promoted a further increase in the fluorescence emission from photosystem I to a level comparable with that obtained at temperatures below 120 degrees K and atmospheric pressure. On the other hand, when the temperature was decreased under pressure, the PBS emission diminished to very low value at blue or green excitation, suggesting the disassembly into the phycobiliprotein subunits.

Biophysical Phenomena↗
PubMed1991

Preparation and characterization of the water-soluble heme-binding domain of cytochrome c1 from the Rhodobacter sphaeroides bc1 complex.

The ubiquinol:cytochrome c2 oxidoreductase (bc1 complex) of Rhodobacter sphaeroides consists of four subunits. One of these subunits, cytochrome c1, is the site of interaction with cytochrome c2, a periplasmic protein. In addition, the sequences of the fbcC gene and of the cytochrome c1 subunit that it encodes suggest that the protein should be located on the periplasmic side of the cytoplasmic membrane and that it is anchored to the membrane by a single membrane-spanning alpha-helix located at the carboxyl-terminal end of the polypeptide. Site-directed mutagenesis of the fbcC gene was used to alter the codon for Gln228 to a stop codon. This results in the production of a truncated version of the cytochrome c1 subunit that lacks the membrane anchor at the carboxyl terminus. The bc1 complex fails to assemble properly as a result of this mutation, but the Rb. sphaeroides cells expressing the altered gene contain a water-soluble form of cytochrome c1 in the periplasm. The water-soluble cytochrome c1 was purified and characterized. The amino-terminal sequence is identical with that of the membrane-bound subunit, indicating the signal sequence is properly processed. High pressure liquid chromatography gel filtration chromatography indicates it is monomeric (28 kDa). The heme content and electrochemical properties are similar to those of the intact subunit within the complex. Flash-induced electron transfer kinetics measured using whole cells demonstrated that the water-soluble cytochrome c1 is competent as a reductant for cytochrome c2 within the periplasmic space. These data show that the isolated water-soluble cytochrome c1 retains many of the properties of the membrane-bound subunit of the bc1 complex and, therefore, will be useful for further structural and functional characterization.

Amino Acid Sequence↗
PubMed1991

Assignment of the histidine axial ligands to the cytochrome bH and cytochrome bL components of the bc1 complex from Rhodobacter sphaeroides by site-directed mutagenesis.

The cytochrome b subunit of the bc1 complex contains two cytochrome components, cytochrome bH and cytochrome bL. Sequence comparisons of this polypeptide from a number of organisms have revealed four invariant histidines which have been postulated to be the heme ligands for the two protoheme IX prosthetic groups. In Rhodobacter sphaeroides, these correspond to His97, His111, His198, and His212. In this paper, the results of amino acid substitutions at each of these positions are reported. Replacement of His97 by either Asp or Asn and of His198 by Asn or Tyr resulted in loss of both cytochrome components. However, His111Asn, His111Asp, and His212Asp all resulted in the selective loss of cytochrome bH and the retention of cytochrome bL. Furthermore, flash kinetics studies show that the myxothiazol-sensitive quinol oxidase (Qz) site associated with cytochrome bL is still functional. These data support the assignment of the axial ligands to cytochrome bH (His111 and His212) and cytochrome bL (His97 and His198). This pairing is consistent with current models of the cytochrome b subunit with eight transmembrane alpha-helices.

Amino Acid Sequence↗
PubMed1991

The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides.

The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.

Alkaline Phosphatase↗
PubMed1990

Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides. Characterization of fbc deletion mutants and complementation by a site-specific mutational variant.

The ubiquinol: cytochrome-c oxidoreductase (cytochrome bc1 complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc1 complex has a dual function. When the cells are grown photosynthetically, the bc1 complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc1 complex is a necessary component of the cytochrome-c2-dependent respiratory chain. The fact that the bc1 complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site-directed mutagenesis to address questions relating to the structure and function of the bc1 complex. In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe-2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc1 complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc1 complex which, as expected, is resistant to the inhibitor. Hence, this system is suitable for the detailed characterization of the bc1 complex, combining site-directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.

Amino Acid Sequence↗
PubMed1990

Large-scale purification and characterization of a highly active four-subunit cytochrome bc1 complex from Rhodobacter sphaeroides.

A highly active, large-scale preparation of ubiquinol:cytochrome c2 oxidoreductase (EC 1.10.2.2; cytochrome bc1 complex) has been obtained from Rhodobacter sphaeroides. The enzyme was solubilized from chromatophores by using dodecyl maltoside in the presence of glycerol and was purified by anion-exchange and gel filtration chromatography. The procedure yields 35 mg of pure bc1 complex from 4.5 g of membrane protein, and its consistently results in an enzyme preparation that catalyzes the reduction of horse heart cytochrome c with a turnover of 250-350 (mumol of cyt c reduced).(mumol of cyt c1)-1.s-1. The turnover number is at least double that of the best preparation reported in the literature [Ljungdahl, P. O., Pennoyer, J. D., Robertson, D. C., & Trumpower, B. L. (1987) Biochim. Biophys. Acta 891, 227-241]. The scale is increased 25-fold, and the yield is markedly improved by using this protocol. Four polypeptide subunits were observed by SDS-PAGE, with Mr values of 40K, 34K, 24K, and 14K. N-Terminal amino acid sequences were obtained for cytochrome c1, the iron-sulfur protein subunit, and for cytochrome b and were identical with the expected protein sequences deduced from the DNA sequence of the fbc operon, with the exceptions that a 22-residue fragment is processed off of the N-terminus of cytochrome c1 and the N-terminal methionine residue is cleaved off both the b cytochrome and iron-sulfur protein subunits. Western blotting experiments indicate that subunit IV is not a contaminating light-harvesting complex polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Phenotypic and genetic characterization of cytochrome c2 deficient mutants of Rhodobacter sphaeroides.

Rhodobacter sphaeroides mutants lacking cytochrome c2 (cyt c2) have been constructed by site-specific recombination between the wild-type genomic cyt c2 structural gene (cycA) and a suicide plasmid containing a defective cyc operon where deletion of cycA sequences was accompanied by insertion of a KnR gene. Southern blot analysis confirmed that the wild-type cyc operon was exchanged for the inactivated cycA gene, presumably by double-reciprocal recombination. Spectroscopic and immunochemical measurements, together with genetic complementation, established that the inability of these mutants to grow under photosynthetic conditions was due to the lack of cyt c2. The cyt c2 deficient strains reduced photooxidized reaction center complexes approximately 4 orders of magnitude more slowly than the parent strain. The phenotype and characteristics of these mutants were restored when a wild-type cyc operon was introduced on a stable low copy number plasmid. These experiments provide the first genetic evidence for the obligatory role of cyt c2 in wild-type cyclic photosynthetic electron transport in R. sphaeroides. We have also observed that the R. sphaeroides cyt c2 deficient strains spontaneously gave rise to photosynthetically competent pseudorevertants at a frequency which suggests that the cyt c2 independent photosynthetic electron transport which suppresses the phenotype of the cyt c2 deficient strains was the result of a single mutation elsewhere in the genome.

Cytochrome c Group↗