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PubMed1992

Rhodobacter capsulatus mutants lacking the Rieske FeS protein form a stable cytochrome bc1 subcomplex with an intact quinone reduction site.

The ubiquinol-cytochrome c oxidoreductase (or bc1 complex) of Rhodobacter capsulatus consists of three subunits: cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein, encoded by the fbcF, fbcB, and fbcC genes, respectively. In the preceding paper [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry (preceding paper in this issue)], we have observed that the apoproteins for cytochromes b and c1 are fully present in the intracytoplasmic membrane of R. capsulatus mutants containing low amounts of, or no, Rieske apoprotein. Here we present evidence that the redox midpoint potentials of cytochromes b and c1, as well as their ability to bind antimycin and stabilize a semiquinone at the Qi site, are unaffected by the absence of the Rieske subunit. This is the first report describing a mutant containing a stable bc1 subcomplex with an intact Qi site in the chromatophore membranes, and provides further evidence that a functional quinone reduction site can be formed in the absence of a quinol oxidation (Qo) site. Additional mutants carrying fbc deletions expressing the remaining subunits of the cytochrome bc1 complex were constructed to investigate the relationship among these subunits for their stability in vivo. Western blot analysis of these mutants indicated that cytochromes b and c1 protect each other against degradation, suggesting that they form a two-protein subcomplex in the absence of the Rieske protein subunit.

Antimycin A↗

PubMed2000

Interactions between the donor and acceptor sides in bacterial reaction centers.

The apparent equilibrium constant K'(2) for electron transfer between the primary (Q(A)) and secondary (Q(B)) quinone acceptors of the reaction center was measured in chromatophores of Rhodobacter capsulatus. In the presence of the oxidized primary donor P(+), we obtained a value of K'(2)(P(+)) approximately 100 at pH 7.2, based on the rates of recombination from P(+)Q(A-) and P(+)Q(B-). K'(2) was also measured in the presence of reduced P, from the damping of semiquinone oscillations during a series of single turnover flashes. A 5-fold smaller value, K'(2)(P) approximately 20, was found. Additional information on the interactions between the donor and acceptor sides was obtained by measuring the shift of the midpoint potential of P caused by the presence of Q(B-) or Q(A-)S (where S indicates the presence of the inhibitor stigmatellin). A stabilization of the oxidized state P(+) was observed in both instances, by 10 mV for Q(B-) and 30 mV for Q(A-)S. The larger stabilization of P(+)Q(A-)S with respect to P(+)Q(B-) does not account for the effect of P(+)/P on K'(2). Analysis of these results indicates that the interactions between P(+)/P and Q(A)/Q(A)(-) are markedly modified depending on the occupancy of the Q(B) pocket by ubiquinone or by stigmatellin. We propose that the large value of K'(2)(P(+)) results essentially from a conformational destabilization of the P(+)Q(A-) state, that is relieved when the proximal site of the Q(B) pocket is occupied by stigmatellin.

Bacteriochlorophylls↗

PubMed1992

Probing the primary donor environment in the histidineM200-->leucine and histidineL173-->leucine heterodimer mutants of Rhodobacter capsulatus by light-induced Fourier transform infrared difference spectroscopy.

Light-induced P+QB-/PQB FTIR difference spectra of reaction centers (RCs) have been obtained from chromatophores lacking light-harvesting B800-850 antenna for Rhodobacter capsulatus wild type (WT) and for the two mutants HisM200-->Leu and HisL173-->Leu. The primary donor (P) in both mutants consists of a bacteriochlorophyll-bacteriopheophytin heterodimer. The most prominent difference between the WT and the mutant spectra is in the 1600-1200-cm-1 region. The WT spectrum displays large positive bands at approximately 1290, 1500-1430, and 1580-1530 cm-1. These three bands are either small or altogether absent in the heterodimer spectra. In addition, both heterodimer spectra compare well with the electrochemically generated BChla+/BChla spectrum [Mäntele, W.G., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472]. These observations indicate that the positive charge is localized on the monomeric BChl in the heterodimers. The overall shape of the ester and keto C = O signals in the BChla+/BChla spectrum is maintained in the in situ spectra although significant differences are observed in the frequency, width, and splitting of the bands. The shape of the signal at 1757/1744 cm-1 in HisL173-->Leu is comparable to the 1751/1737-cm-1 signal of BChla+/BChla in tetrahydrofuran, indicating a free 10a ester C = O of PM in HisL173-->Leu. The reduced amplitude of the negative 1740-cm-1 feature in both HisM200-->Leu and WT spectra suggests a hydrogen-bonded 10a ester C = O for PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriochlorophylls↗

PubMed1992

Mutagenesis of methionine-183 drastically affects the physicochemical properties of cytochrome c1 of the bc1 complex of Rhodobacter capsulatus.

Site-directed mutagenesis was used to investigate which of the highly conserved methionine residues (M183 and M205) provides the sixth axial ligand to the heme Fe in the cyt c1 subunit of the bc1 complex from the bacterium Rhodobacter capsulatus. These residues were changed to leucine (cM183L) and valine (cM205V). Two additional mutants were constructed, 1 in which a stop codon was inserted at M205 (cM205*) and the second in which 127 amino acids were deleted between the signal sequence and the putative C-terminal transmembrane alpha-helix (c delta SfuI). Only cM205V grew photosynthetically, and membranes isolated from this strain catalyzed quinol-dependent reduction of cyt c in amounts similar to that in a wild-type strain. Even though cM183L could not grow photosynthetically, it contained all the appropriate polypeptides and cofactors of the bc1 complex, as shown by SDS-PAGE and optical difference spectroscopy of intact membrane particles. Neither of the two deletion mutants contained a stable complex. Flash absorption spectroscopy using chromatophores showed no cytochrome c rereduction after oxidation by the reaction center in cM183L. The bc1 complex from each strain was isolated and characterized. Oxidation reduction midpoint potential titrations revealed that cyt c1 from cM183L had a dramatically shifted Em value (delta Em = -390 mV) compared with wild type and cM205V. While the optical absorption spectrum of cyt c1 from cM183L suggested that the c-type heme was low-spin, nonetheless it was able to react with the exogenous ligand carbon monoxide. The overall data support that M183, and not M205, is the sixth ligand to the heme Fe of cyt c1 of the bc1 complex.

Amino Acid Sequence↗

PubMed1993

Structure, spectroscopic, and redox properties of Rhodobacter sphaeroides reaction centers bearing point mutations near the primary electron donor.

Single mutations of three amino acid residues in the vicinity of the primary electron donor, P, in the reaction center (RC) from Rhodobacter (Rb.) sphaeroides were constructed and characterized in order to study the effects of hydrogen-bonding on the physical properties of P. The mutations, Phe M197-->Tyr, Met L248-->Thr, and Ser L244-->Gly, represent single amino acid changes near P designed to introduce residues found in Rhodopseudomonas (Rps.) viridis and to, thus, probe the effects of nonconserved residues. The mutations were designed to change the nonconserved H-bonding interactions of P in Rb. sphaeroides, at the level of a C2 acetyl, a C9 keto, and a C10 ester carbonyl of P, respectively, to those present in Rps. viridis. The Fourier transform (pre)resonance Raman (FTRR) spectra of P, in its reduced and oxidized states, from reaction centers of these mutants were studied to determine modifications of H-bond interactions of the pi-conjugated C2 acetyl and C9 keto carbonyl groups and the C10 carbomethoxy ester carbonyl groups of P. The vibrational spectra of reduced P in the Met L248-->Thr and Ser L244-->Gly mutants reveal no evidence for changes in the H-bonding pattern of P; this suggests that for Rb. sphaeroides wild type, Ser L244 is not H-bonded to the C10 ester carbonyl of PL. The vibrational spectrum of reduced P from the Phe M197-->Tyr mutant compared to that of wild type can unambiguously be interpreted in terms of the formation of a new H-bond with an acetyl carbonyl of P, specifically PM. Correlating with the new H-bond, the Phe M197-->Tyr mutant exhibits an electronic absorption spectrum where the P absorption band is significantly perturbed. Intact cell and chromatophore photobleaching spectra of the same mutant indicate that the P absorption band has red-shifted by ca. 10 nm; no such behavior is observed for the other mutants. As well, the P-->BPheL electron transfer rate does not seem to strongly depend on the H-bonding of the C2 acetyl carbonyl of PM to a tyrosine residue. The EPR zero-field splitting parameters, E and D, of the primary donor triplet are only slightly modified in the mutant reaction centers, on the order of 1%.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriochlorophylls↗

PubMed1991

Involvement of the protein-protein interactions in the thermodynamics of the electron-transfer process in the reaction centers from Rhodopseudomonas viridis.

Reaction centers from Rhodopseudomonas viridis were reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) liposomes. Freeze-fracture electron micrographs were performed on the samples frozen from temperatures above and below the phase transition temperatures of those lipids (Tc = 23 and 9.5 degrees C, in DMPC and DEPC, respectively). Above Tc, in the fluid conformation of the lipids, the reaction centers are randomly distributed in the vesicle membranes. Below Tc, aggregation of the proteins occurs. The Arrhenius plots of the rate constants of the charge recombination between P+ and QA- display a break at about 24 degrees C in DMPC vesicles and about 10 degrees C in DEPC vesicles (P represents the primary electron donor, a dimer of bacteriochlorophyll, and QA the primary quinone electron acceptor). This is in contrast to what was previously observed for the proteoliposomes of egg yolk phosphatidylcholine and for chromatophores [Baciou, L., Rivas, E., & Sebban, P. (1990) Biochemistry 29, 2966-2976], for which Arrhenius plots were linear. In DMPC and DEPC proteoliposomes, the activation parameters were very different on the two sides of Tc (delta H degrees for T less than Tc = 2.5 times delta H degrees for T greater than Tc), leading however, to the same delta G degrees values. Taking into account the structural and thermodynamic data, we suggest that, in vivo, protein-protein interactions play a role in the thermodynamic parameters associated with the energy stabilization process within the reaction centers.

Dimyristoylphosphatidylcholine↗

PubMed1988

Reconstitution of the B873 light-harvesting complex of Rhodospirillum rubrum from the separately isolated alpha- and beta-polypeptides and bacteriochlorophyll a.

The light-harvesting complex of Rhodospirillum rubrum was reversibly dissociated into its component parts: bacteriochlorophyll and two 6-kilodalton polypeptides. The dissociation of the complex by n-octyl beta-D-glucopyranoside was accompanied by a shift of the absorbance maximum from 873 to 820 nm (a stable intermediate form) and finally to 777 nm. In the latter state, bacteriochlorophyll was shown to be free from the protein. Complexes absorbing at 820 and 873 nm could be re-formed from the fully dissociated state with over 80% yield by dilution of the detergent. Absorbance and circular dichroism properties of the re-formed B820 complex were essentially identical with those of B820 formed from chromatophores. Phospholipids and higher concentrations of complex were required to obtain the in vivo circular dichroism spectrum for reassociated B873. Reconstitution of the light-harvesting complexes from separately isolated alpha- and beta-polypeptides and bacteriochlorophyll was also demonstrated. Absorbance and circular dichroism spectra of these complexes were identical with those of complexes formed by the reassociation of the dissociated complex. Bacteriochlorophyll and the beta-polypeptide alone formed a complex that had an absorbance at 820 nm, but an 873-nm complex could not be formed without addition of the alpha-polypeptide. The alpha-polypeptide alone with bacteriochlorophyll did not form any red-shifted complex. In preliminary structure-function studies, some analogues of bacteriochlorophyll were also tested for reconstitution.

Bacterial Proteins↗

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↗

PubMed1990

EPR characterization of genetically modified reaction centers of Rhodobacter capsulatus.

Electron paramagnetic resonance (EPR) has been used to investigate the cation and triplet states of Rhodobacter capsulatus reaction centers (RCs) containing amino acid substitutions affecting the primary donor, monomeric bacteriochlorophylls (Bchls), and the photoactive bacteriopheophytin (Bphe). The broadened line width of the cation radical in HisM200----Leu and HisM200----Phe reaction centers, whose primary donor consists of a Bchl-Bphe heterodimer, indicates a highly asymmetric distribution of the unpaired electron over the heterodimer. A T0 polarized triplet state with reduced yield is observed in heterodimer-containing RCs. The zero field splitting parameters indicate that this triplet essentially resides on the Bchl half of the heterodimer. The cation and triplet states of reaction centers containing HisM200----Gln, HisL173----Gln, GluL104----Gln, or GluL104----Leu substitutions are similar to those observed in wild type. Oligonucleotide-mediated mutagenesis has been used to change the histidine residues that are positioned near the central Mg2+ ions of the reaction center monomeric bacteriochlorophylls. Reaction centers containing serine substitutions at M180 and L153 or a threonine substitution at L153 have unaltered pigment compositions and are photochemically active. The cation and triplet states of HisL153----Leu reaction centers are similar to those observed in wild type. Triplet energy transfer to carotenoid is not observed at 100 K in HisM180----Arg chromatophores. These results have important implications for the structural requirements of tetrapyrrole binding and for our understanding of the mechanisms of primary electron transfer in the reaction center.

Bacteriochlorophylls↗

PubMed2002

Modulation of the midpoint potential of the [2Fe-2S] Rieske iron sulfur center by Qo occupants in the bc1 complex.

Following addition of myxothiazol to antimycin-treated chromatophores from Rhodobacter sphaeroides poised at an ambient redox potential (E(h)) of approximately 300 mV, the amplitude of the flash-induced cytochrome c(1) oxidation in the ms range increased, indicating a decrease in the availability of electrons from the immediate donor to c(1), the Rieske iron-sulfur protein (ISP). Because the effect was seen only over the limited E(h) range, we conclude that it is due to a decrease in the apparent midpoint redox potential (E(m)) of the ISP by about 40 mV on addition of myxothiazol. This is in line with the change in E(m) previously seen in direct redox titrations. Our results show that the reduced ISP binds with quinone at the Q(o) site with a higher affinity than does the oxidized ISP. The displacement of ubiquinone by myxothiazol leads to elimination of this preferential binding of the ISP reduced form and results in a shift in the midpoint potential of ISP to a more negative value. A simple hypothesis to explain this effect is that myxothiazol prevents formation of hydrogen bond of ubiquinone with the reduced ISP. We conclude that all Q(o) site occupants (ubiquinone, UHDBT, stigmatellin) that form hydrogen bonds with the reduced ISP shift the apparent E(m) of the ISP in the same direction to more positive values. Inhibitors that bind in the domain of the Q(o) site proximal to heme b(L) (myxothiazol, MOA-stilbene) and displace ubiquinone from the site cause a decrease in E(m) of ISP. We present a new formalism for treatment of the relation between E(m) change and the binding constants involved, which simplifies analysis. Using this formalism, we estimated that binding free energies for hydrogen bond formation with the Q(o) site occupant, range from the largest value of approximately 23 kJ mol(-1) in the presence of stigmatellin (appropriate for the buried hydrogen bond shown by structures), to a value of approximately 3.5 kJ mol(-1) in the native complex. We discuss this range of values in the context of a model in which the native structure constrains the interaction of ISP with the Q(o) site occupant so as to favor dissociation and the faster kinetics of unbinding necessary for rapid turnover.

Binding Sites↗

PubMed2004

Light-harvesting complex 1 stabilizes P+QB- charge separation in reaction centers of Rhodobacter sphaeroides.

The kinetics of charge recombination following photoexcitation by a laser pulse have been analyzed in the reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides. In RC-LH1 core complexes isolated from photosynthetically grown cells P(+)Q(B)(-) recombines with an average rate constant, k approximately 0.3 s(-1), more than three times smaller than that measured in RC deprived of the LH1 (k approximately 1 s(-1)). A comparable, slowed recombination kinetics is observed in RC-LH1 complexes purified from a pufX-deleted strain. Slowing of the charge recombination kinetics is even more pronounced in RC-LH1 complexes isolated from wild-type semiaerobically grown cells (k approximately 0.2 s(-1)). Since the kinetics of P(+)Q(A)(-) recombination is unaffected by the presence of the antenna, the P(+)Q(B)(-) state appears to be energetically stabilized in core complexes. Determinations of the ubiquinone-10 (UQ(10)) complement associated with the purified RC-LH1 complexes always yield UQ(10)/RC ratios larger than 10. These quinone molecules are functionally coupled to the RC-LH1 complex, as judged from the extent of exogenous cytochrome c(2) rapidly oxidized under continuous light excitation. Analysis of P(+)Q(B)(-) recombination, based on a kinetic model which considers fast quinone equilibrium at the Q(B) binding site, indicates that the slowing down of charge recombination kinetics observed in RC-LH1 complexes cannot be explained solely by a quinone concentration effect and suggests that stabilization of the light-induced charge separation is predominantly due to interaction of the Q(B) site with the LH1 complex. The high UQ(10) complements detected in RC-LH1 core complexes, but not in purified light-harvesting complex 2 and in RC, are proposed to reflect an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer.

Bacterial Proteins↗

PubMed2006

High yield of M-side electron transfer in mutants of Rhodobacter capsulatus reaction centers lacking the L-side bacteriopheophytin.

We present studies on a series of photosynthetic reaction center (RC) mutants created in the background of the Rhodobacter capsulatus D(LL) mutant, in which the D helix of the M subunit has been substituted with that from the L subunit. Previous work on the D(LL) mutant in chromatophore preparations showed that RCs assembled without the bacteriopheophytin H(L) electron acceptor and performed no charge separation following light absorption. We have successfully isolated poly-His-tagged D(LL) RCs by using the detergent Deriphat 160-C and shown that the RCs are devoid of H(L). The excited state of the primary electron donor, P*, is found to have a lifetime of 180 +/- 20 ps and to decay exclusively (>95%) via internal conversion to the ground state, with no evidence for formation of any charge-separated intermediates. By additional mutation in the D(LL) background of two residues that affect the P/P+ oxidation potential and one that facilitates M-side electron transfer, we achieve an unprecedented 70% yield of P+ H(M)-, more than doubling the highest yield of this state achieved previously. This result underscores the importance of the relative free energies of P* and the charge-separated states in governing the rates and yields of electron transfer in bacterial RCs and provides a basis for systematically investigating M-side electron transfer without any competition from the native L-side pathway.

Amino Acid Sequence↗

PubMed2006

In situ kinetics of cytochromes c1 and c2.

In Rhodobacter sphaeroides chromatophores, cytochromes (cyt) c(1) and c(2) have closely overlapping spectra, and their spectral deconvolution provides a challenging task. As a result, analyses of the kinetics of different cytochrome components of the bc(1) complex in purple bacteria usually report only the sum cyt c(1) + cyt c(2) kinetics. Here we used newly determined difference spectra of individual components to resolve the kinetics of cyt c(1) and c(2) in situ via a least-squares (LS) deconvolution. We found that the kinetics of cyt c(1) and c(2) are significantly different from those measured using the traditional difference wavelength (DW) approach, based on the difference in the absorbance at two different wavelengths specific for each component. In particular, with the wavelength pairs previously recommended, differences in instrumental calibration led to kinetics of flash-induced cyt c(1) oxidation measured with the DW method which were faster than those determined by the LS method (half-time of approximately 120 micros vs half-time of approximately 235 micros, in the presence of antimycin). In addition, the LS approach revealed a delay of approximately 50 micros in the kinetics of cyt c(1) oxidation, which was masked when the DW approach was used. We attribute this delay to all processes leading to the oxidation of cyt c(1) after light activation of the photosynthetic reaction center, especially the dissociation of cyt c(2) from the reaction center. We also found that kinetics of both cyt c(1) and c(2) measured by the DW approach were significantly distorted at times longer than 1 ms, due to spectral contamination from changes in the b hemes. The successful spectral deconvolution of cyt c(1) and c(2), and inclusion of both cytochromes in the kinetic analysis, significantly increase the data available for mechanistic understanding of bc(1) turnover in situ.

Antimycin A↗

PubMed2006

Functional and structural analysis of the photosynthetic apparatus of Rhodobacter veldkampii.

In the widely studied purple bacterium Rhodobacter sphaeroides, a small transmembrane protein, named PufX, is required for photosynthetic growth and is involved in the supramolecular dimeric organization of the core complex. We performed a structural and functional analysis of the photosynthetic apparatus of Rhodobacter veldkampii, a related species which evolved independently. Time-resolved optical spectroscopy of R. veldkampii chromatophores showed that the reaction center shares with R. sphaeroides spectral and redox properties and interacts with a cytochrome bc(1) complex through a Q-cycle mechanism. Kinetic analysis of flash-induced cytochrome b(561) reduction indicated a fast delivery of the reduced quinol produced by the reaction center to the cytochrome bc(1) complex. A core complex, along with two light-harvesting LH2 complexes significantly different in size, was purified and analyzed by sedimentation, size exclusion chromatography, mass spectroscopy, and electron microscopy. A PufX subunit identified by MALDI-TOF was found to be associated with the core complex. However, as shown by sedimentation and single-particle analysis by electron microscopy, the core complex is monomeric, suggesting that in R. veldkampii, PufX is involved in the photosynthetic growth but is unable to induce the dimerization of the core complex.

Amino Acid Sequence↗

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↗

PubMed1997

Site-directed mutations near the L-subunit D-helix of the purple bacterial reaction center: a partial model for the primary donor of photosystem II.

We have engineered a photosynthetically competent mutant of the purple non-sulfur bacterium Rhodobacter capsulatus which seeks to mimic the behavior of the primary electron donor (P) of the plant photosystem II (PS II) reaction center (RC). To construct this mutant (denoted D1-ILMH), four residues in the bacterial L subunit were mutagenized, such that an 11-residue segment was made identical to the analogous segment from the D1 subunit of PS II. The electronic properties of the bacteriochlorophyll (Bchl) dimer which constitutes the primary donor are substantially altered by these modifications, to the degree that the dimer becomes functionally much more "monomeric". The changes include (1) an increase in the values of the zero-field splitting (ZFS) parameters, as measured by electron paramagnetic resonance (EPR), for the spin-polarized triplet state, 3P, from /D/ = 185 x 10(-4) cm(-1) and /E/ = 31 x 10(-4) cm(-1) in wild-type (WT) chromatophore membranes to /D/ = 200 x 10(-4) cm(-1) and /E/ = 44 x 10(-4) cm(-1) in the mutant and (2) an increase in the EPR line width of the oxidized state, P+, from 0.97 mT in WT to 1.09 mT in D1-ILMH RCs. However, unlike the PS II primary donor (P680), the orientation of 3P in the D1-ILMH mutant is the same as in WT bacteria and does not display the unusual orientation found for PS II. And whereas the redox couple P/P+ has a very high midpoint potential in PS II, P/P+ in the D1-ILMH mutant has a lower midpoint (90 mV more negative) than in WT Rb. capsulatus. In addition, Raman measurements indicate that the hydrogen bond between HisL168 and the C2 acetyl carbonyl oxygen of the Bchl on the active electron transfer pathway (P(A)) is absent in the mutant, due to the fact that HisL168 in the WT sequence has been replaced by a leucine in D1-ILMH. However, the Raman data also reveal the presence of a new hydrogen bond in the D1-ILMH RCs, between the C9 keto carbonyl oxygen of P(A) and an unknown hydrogen-bond donor. Thus, although the protein environment around one of the Bchls of the special pair is significantly changed in D1-ILMH, the chimeric RC does not, as a result of these changes, have a primary donor that is oriented like the one in PS II.

Amino Acid Sequence↗

PubMed1997

Two distinct conformations of the primary electron donor in reaction centers from Rhodobacter sphaeroides revealed by ENDOR/TRIPLE-spectroscopy.

The effect of solubilization of photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides with different detergents on the electronic structure of the oxidized primary donor, P.+, is investigated. Electron paramagnetic resonance spectroscopy and related multiple resonance techniques (ENDOR/Special TRIPLE) show that two distinct conformations of P.+ can be obtained, depending on the detergent properties, the detergent/RC ratio, and the temperature. The two states correspond to different positions of the long-wavelength Qy-band of the neutral state, P (lambda1 = 866 nm and lambda2 = 850 nm at room temperature) and therefore are called P866.+ and P850.+, respectively. P866.+ is found in chromatophores and in RCs solubilized with nonionic detergents and bile salts. P850.+ is induced by zwitterionic and ionic detergents with aliphatic hydrophobic chains. The TRIPLE resonance spectra reveal that both states coexist in the range lambda2 < lambda(max) < lambda1. The main property of the detergent that determines the ability to induce P850.+ is the polarity of the head group. A simple phenomenological model is presented that relates the standard Gibbs free energy difference between the two conformations to the detergent/RC ratio and the temperature. Of special interest is the observation that the widely used detergent LDAO can induce P850.+ upon freezing the RCs without cryoprotectants. The spectroscopic properties of the two states are compared and their possible roles in RC function are discussed.

Bile Acids and Salts↗

PubMed1998

Isolation and characterization of a two-subunit cytochrome b-c1 subcomplex from Rhodobacter capsulatus and reconstitution of its ubihydroquinone oxidation (Qo) site with purified Fe-S protein subunit.

The presence of a two-subunit cytochrome (cyt) b-c1 subcomplex in chromatophore membranes of Rhodobacter capsulatus mutants lacking the Rieske iron-sulfur (Fe-S) protein has been described previously [Davidson, E., Ohnishi, T., Tokito, M., and Daldal, F. (1992) Biochemistry 31, 3351-3358]. Here, this subcomplex was purified to homogeneity in large quantities, and its properties were characterized. As expected, it contained stoichiometric amounts of cyt b and cyt c1 subunits forming a stable entity devoid of the Fe-S protein subunit. The spectral and thermodynamic properties of its heme groups were largely similar to those of a wild-type bc1 complex, except that those of its cyt bL heme were modified as revealed by EPR spectroscopy. Dark potentiometric titrations indicated that the redox midpoint potential (Em7) values of cytochromes bH, bL, and c1 were very similar to those of a wild-type bc1 complex. The purified b-c1 subcomplex had a nonfunctional ubihydroquinone (UQH2) oxidation (Qo) site, but it contained an intact ubiquinone (UQ) reductase (Qi) site as judged by its ability to bind the Qi inhibitor antimycin A, and by the presence of antimycin A sensitive Qi semiquinone. Interestingly, its Qo site could be readily reconstituted by addition of purified Fe-S protein subunit. Reactivated complex exhibited myxothiazol, stigmatellin, and antimycin A sensitive cyt c reductase activity and an EPR gx signal comparable to that observed with a bc1 complex when the Qo site is partially occupied with UQ/UQH2. However, a mutant derivative of the Fe-S protein subunit lacking its first 43 amino acid residues was unable to reactivate the purified b-c1 subcomplex although it could bind to its Qo site in the presence of stigmatellin. These findings demonstrated for the first time that the amino-terminal membrane-anchoring domain of the Fe-S protein subunit is necessary for UQH2 oxidation even though its carboxyl-terminal domain is sufficient to provide wild-type-like interactions with stigmatellin at the Qo site of the bc1 complex.

Binding Sites↗