Cytochromes C Research Articles

Zinc Deficiency Impacts CO2 Assimilation and Disrupts Copper Homeostasis in Chlamydomonas reinhardtii

Zinc is an essential nutrient because of its role in catalysis and in protein stabilization, but excess zinc is deleterious. We distinguished four nutritional zinc states in the alga Chlamydomonas reinhardtii: toxic, replete, deficient, and limited. Growth is inhibited in zinc-limited and zinc-toxic cells relative to zinc-replete cells, whereas zinc deficiency is visually asymptomatic but distinguished by the accumulation of transcripts encoding ZIP family transporters. To identify targets of zinc deficiency and mechanisms of zinc acclimation, we used RNA-seq to probe zinc nutrition-responsive changes in gene expression. We identified genes encoding zinc-handling components, including ZIP family transporters and candidate chaperones. Additionally, we noted an impact on two other regulatory pathways, the carbon-concentrating mechanism (CCM) and the nutritional copper regulon. Targets of transcription factor Ccm1 and various CAH genes are up-regulated in zinc deficiency, probably due to reduced carbonic anhydrase activity, validated by quantitative proteomics and immunoblot analysis of Cah1, Cah3, and Cah4. Chlamydomonas is therefore not able to grow photoautotrophically in zinc-limiting conditions, but supplementation with 1% CO2 restores growth to wild-type rates, suggesting that the inability to maintain CCM is a major consequence of zinc limitation. The Crr1 regulon responds to copper limitation and is turned on in zinc deficiency, and Crr1 is required for growth in zinc-limiting conditions. Zinc-deficient cells are functionally copper-deficient, although they hyperaccumulate copper up to 50-fold over normal levels. We suggest that zinc-deficient cells sequester copper in a biounavailable form, perhaps to prevent mismetallation of critical zinc sites.

A critical role for the cccA gene product, cytochrome c2, in diverting electrons from aerobic respiration to denitrification in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a microaerophile that, when oxygen availability is limited, supplements aerobic respiration with a truncated denitrification pathway, nitrite reduction to nitrous oxide. We demonstrate that the cccA gene of Neisseria gonorrhoeae strain F62 (accession number NG0292) is expressed, but the product, cytochrome c2, accumulates to only low levels. Nevertheless, a cccA mutant reduced nitrite at about half the rate of the parent strain. We previously reported that cytochromes c4 and c5 transfer electrons to cytochrome oxidase cbb3 by two independent pathways and that the CcoP subunit of cytochrome oxidase cbb3 transfers electrons to nitrite. We show that mutants defective in either cytochrome c4 or c5 also reduce nitrite more slowly than the parent. By combining mutations in cccA (Δc2), cycA (Δc4), cycB (Δc5), and ccoP (ccoP-C368A), we demonstrate that cytochrome c2 is required for electron transfer from cytochrome c4 via the third heme group of CcoP to the nitrite reductase, AniA, and that cytochrome c5 transfers electrons to nitrite reductase by an independent pathway. We propose that cytochrome c2 forms a complex with cytochrome oxidase. If so, the redox state of cytochrome c2 might regulate electron transfer to nitrite or oxygen. However, our data are more consistent with a mechanism in which cytochrome c2 and the CcoQ subunit of cytochrome oxidase form alternative complexes that preferentially catalyze nitrite and oxygen reduction, respectively. Comparison with the much simpler electron transfer pathway for nitrite reduction in the meningococcus provides fascinating insights into niche adaptation within the pathogenic neisseriae.

Nano-mechanical mapping of the interactions between surface-bound RC-LH1-PufX core complexes and cytochrome c 2 attached to an AFM probe

Electron transfer pathways in photosynthesis involve interactions between membrane-bound complexes such as reaction centres with an extrinsic partner. In this study, the biological specificity of electron transfer between the reaction centre-light-harvesting 1-PufX complex and its extrinsic electron donor, cytochrome c2, formed the basis for mapping the location of surface-attached RC-LH1-PufX complexes using atomic force microscopy (AFM). This nano-mechanical mapping method used an AFM probe functionalised with cyt c2 molecules to quantify the interaction forces involved, at the single-molecule level under native conditions. With surface-bound RC-His12-LH1-PufX complexes in the photo-oxidised state, the mean interaction force with cyt c2 is approximately 480 pN with an interaction frequency of around 66 %. The latter value lowered 5.5-fold when chemically reduced RC-His12-LH1-PufX complexes are imaged in the dark to abolish electron transfer from cyt c2 to the RC. The correspondence between topographic and adhesion images recorded over the same area of the sample shows that affinity-based AFM methods are a useful tool when topology alone is insufficient for spatially locating proteins at the surface of photosynthetic membranes.

The Heme Chaperone ApoCcmE Forms a Ternary Complex with CcmI and Apocytochrome c

Cytochrome c maturation (Ccm) is a post-translational process that occurs after translocation of apocytochromes c to the positive (p) side of energy-transducing membranes. Ccm is responsible for the formation of covalent bonds between the thiol groups of two cysteines residues at the heme-binding sites of the apocytochromes and the vinyl groups of heme b (protoporphyrin IX-Fe). Among the proteins (CcmABCDEFGHI and CcdA) required for this process, CcmABCD are involved in loading heme b to apoCcmE. The holoCcmE thus formed provides heme b to the apocytochromes. Catalysis of the thioether bonds between the apocytochromes c and heme b is mediated by the heme ligation core complex, which in Rhodobacter capsulatus contains at least the CcmF, CcmH, and CcmI components. In this work we show that the heme chaperone apoCcmE binds to the apocytochrome c and the apocytochrome c chaperone CcmI to yield stable binary and ternary complexes in the absence of heme in vitro. We found that during these protein-protein interactions, apoCcmE favors the presence of a disulfide bond at the apocytochrome c heme-binding site. We also establish using detergent-dispersed membranes that apoCcmE interacts directly with CcmI and CcmH of the heme ligation core complex CcmFHI. Implications of these findings are discussed with respect to heme transfer from CcmE to the apocytochromes c during heme ligation assisted by the core complex CcmFHI.

Two types of fucoxanthin-chlorophyll-binding proteins I tightly bound to the photosystem I core complex in marine centric diatoms

Intact fucoxanthin (Fucox)-chlorophyll (Chl)-binding protein I-photosystem I supercomplexes (FCPI-PSIs) were prepared by a newly developed simple fast procedure from centric diatoms Chaetoceros gracilis and Thalassiosira pseudonana to study the mechanism of their efficient solar energy accumulation. FCPI-PSI purified from C. gracilis contained 252 Chl a, 23 Chl c, 56 Fucox, 34 diadinoxanthin+diatoxanthin, 1 violaxanthin, 21 ß-carotene, and 2 menaquinone-4 per P700. The complex showed a high electron transfer activity at 185,000μmolmg Chl a−1·h−1 to reduce methyl viologen from added cytochrome c6. We identified 14 and 21 FCP proteins in FCPI-PSI of C. gracilis and T. pseudonana, respectively, determined by N-terminal and internal amino acid sequences and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analyses. PsaO and a red lineage Chla/b-binding-like protein (RedCAP), Thaps3:270215, were also identified. Severe detergent treatment of FCPI-PSI released FCPI-1 first, leaving the FCPI-2-PSI-core complex. FCPI-1 contained more Chl c and showed Chl a fluorescence at a shorter wavelength than FCPI-2, suggesting an excitation-energy transfer from FCPI-1 to FCPI-2 and then to the PSI core. Fluorescence emission spectra at 17K in FCPI-2 varied depending on the excitation wavelength, suggesting two independent energy transfer routes. We formulated a model of FCPI-PSI based on the biochemical assay results.

Electron transfer between multihaem cytochromes c3 from Desulfovibrio africanus

The tetrahaem type I cytochromes c3 from Desulfovibrionaceae shuttle electrons from a periplasmic hydrogenase to transmembrane electron transfer complexes. In D. africanus, it is believed that the electrons are received by another tetrahaem cytochrome c3, denoted type II, which is associated with the membrane complex. Thermodynamic measurements show that the type I cytochrome c3 has the potential to transfer two electrons at a time. This study uses two-dimensional NMR to investigate the exchange of electrons between type I and type II cytochromes c3 at equilibrium in intermediate stages of oxidation. The results indicate that the two proteins are physiological partners but that only single-electron transfers occur in solution.

Fractionation of sulfur isotopes by Desulfovibrio vulgaris mutants lacking hydrogenases or type I tetraheme cytochrome c3

The sulfur isotope effect produced by sulfate reducing microbes is commonly used to trace biogeochemical cycles of sulfur and carbon in aquatic and sedimentary environments. To test the contribution of intracellular coupling between carbon and sulfur metabolisms to the overall magnitude of the sulfur isotope effect, this study compared sulfur isotope fractionations by mutants of Desulfovibrio vulgaris Hildenborough. We tested mutant strains lacking one or two periplasmic (Hyd, Hyn-1, Hyn-2, and Hys) or cytoplasmic hydrogenases (Ech and CooL), and a mutant lacking type I tetraheme cytochrome (TpI-c3). In batch culture, wild-type D. vulgaris and its hydrogenase mutants had comparable growth kinetics and produced the same sulfur isotope effects. This is consistent with the reported redundancy of hydrogenases in D. vulgaris. However, the TpI-c3 mutant (ΔcycA) exhibited slower growth and sulfate reduction rates in batch culture, and produced more H2 and an approximately 50% larger sulfur isotope effect, compared to the wild type. The magnitude of sulfur isotope fractionation in the CycA deletion strain, thus, increased due to the disrupted coupling of the carbon oxidation and sulfate reduction pathways. In continuous culture, wild-type D. vulgaris and the CycA mutant produced similar sulfur isotope effects, underscoring the influence of environmental conditions on the relative contribution of hydrogen cycling to the electron transport. The large sulfur isotope effects associated with the non-ideal stoichiometry of sulfate reduction in this study imply that simultaneous fermentation and sulfate reduction may be responsible for some of the large naturally-occurring sulfur isotope effects. Overall, mutant strains provide a powerful tool to test the effect of specific redox proteins and pathways on sulfur isotope fractionation.

Fluorescence Assays for Photosynthetic Capacity of Bacteria

The fast kinetics of induction and relaxation of bacteriochlorophyll prompt and delayed fluorescence together with absorption changes of the reaction center (RC) dimer (P) were measured by combination of flashes from laser diodes in intact cells of wild type, carotenoidless (R-26) and cytochrome c2 deficient (CYCA) mutants of photosynthetic bacteria Rhodobacter sphaeroides. The fluorescence induction under high intensity of continuous light splits into fast and slow rises both overlapped by the (carotenoid and/or bacteriochlorophyll) triplet quenching. The fast phase is purely photochemical as it depends strongly on the number of photons absorbed. The slow phase is the combination of thermal and photochemical reactions and reflects the multiple turnover of the system. Upon short flash, the fluorescence yield cannot reach the maximum due to partial reopening of the RCs by rapid donor and acceptor side reactions. Longer flashes are needed to close the RC completely. Contrary to higher plants, the kinetics of induction and relaxation of the fluorescence yield in bacteria are controlled principally by P+. The reactions on the quinone side play minor role. The quantitative determination of the cyclic electron transfer rate can be based on calibration to the quantity of P+.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Molecular dynamics study on binding free energy of Azurin–Cytochrome c551 complex

The thermodynamic stability and binding characteristics of Azurin–Cytochrome c551 (AZ/C551) complex in water solvent were investigated by an all-atom molecular dynamics (MD) simulation. Model complexes of AZ/C551 were prepared by protein–protein docking and MD simulations. The total free energy and binding free energy of model AZ/C551 complexes were evaluated using the conformational and solvation energies and entropy terms. The conformational and solvation free energies were found to largely depend on the hydrophilicity/hydrophobicity of the AZ/C551 complex, showing the importance of these characteristics in the thermodynamic stability and in the formation of the AZ/C551 complex.

Mind the gap: cytochrome interactions reveal electron pathways across the periplasm ofShewanella oneidensisMR-1

Extracellular electron transfer is the key metabolic trait that enables some bacteria to play a significant role in the biogeochemical cycling of metals and in bioelectrochemical devices such as microbial fuel cells. In Shewanella oneidensis MR-1, electrons generated in the cytoplasm by catabolic processes must cross the periplasmic space to reach terminal oxidoreductases found at the cell surface. Lack of knowledge on how these electrons flow across the periplasmic space is one of the unresolved issues related with extracellular electron transfer. Using NMR to probe protein-protein interactions, kinetic measurements of electron transfer and electrostatic calculations, we were able to identify protein partners and their docking sites, and determine the dissociation constants. The results showed that both STC (small tetrahaem cytochrome c) and FccA (flavocytochrome c) interact with their redox partners, CymA and MtrA, through a single haem, avoiding the establishment of stable redox complexes capable of spanning the periplasmic space. Furthermore, we verified that the most abundant periplasmic cytochromes STC, FccA and ScyA (monohaem cytochrome c5) do not interact with each other and this is likely to be the consequence of negative surface charges in these proteins. This reveals the co-existence of two non-mixing redox pathways that lead to extracellular electron transfer in S. oneidensis MR-1 established through transient protein interactions.

The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides

We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. Mössbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θD0~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c2, and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.

The Conserved His-144 in the PsbP Protein Is Important for the Interaction between the PsbP N-terminus and the Cyt b559 Subunit of Photosystem II

The PsbP protein regulates the binding properties of Ca(2+) and Cl(-), and stabilizes the Mn cluster of photosystem II (PSII); however, the binding site and topology in PSII have yet to be clarified. Here we report that the structure around His-144 and Asp-165 in PsbP, which is suggested to be a metal binding site, has a crucial role for the functional interaction between PsbP and PSII. The mutated PsbP-H144A protein exhibits reduced ability to retain Cl(-) anions in PSII, whereas the D165V mutation does not affect PsbP function. Interestingly, H144A/D165V double mutation suppresses the effect of H144A mutation, suggesting that these residues have a role other than metal binding. FTIR difference spectroscopy suggests that H144A/D165V restores proper interaction with PSII and induces the conformational change around the Mn cluster during the S(1)/S(2) transition. Cross-linking experiments show that the H144A mutation affects the direct interaction between PsbP and the Cyt b(559) α subunit of PSII (the PsbE protein). However, this interaction is restored in the H144A/D165V mutant. In the PsbP structure, His-144 and Asp-165 form a salt bridge. H144A mutation is likely to disrupt this bridge and liberate Asp-165, inhibiting the proper PsbP-PSII interaction. Finally, mass spectrometric analysis has identified the cross-linked sites of PsbP and PsbE as Ala-1 and Glu-57, respectively. Therefore His-144, in the C-terminal domain of PsbP, plays a crucial role in maintaining proper N terminus interaction. These data provide important information about the binding characteristics of PsbP in green plant PSII.

An expression system for regulated protein production in Synechocystis sp. PCC 6803 and its application for construction of a conditional knockout of the ferrochelatase enzyme

Synechocystis sp. PCC 6803 is a model organism for the study of photosynthetic processes. Methods to genetically manipulate this bacterium are essential to investigate these processes and to evaluate potential biotechnological applications. We developed a vector for controllable expression of proteins using a platform for stable integration of the expression cassette into the genome. The respective gene is translationally fused to the promoter of the petJ gene encoding cytochrome c553 that is repressed by copper. Maximal expression from this promoter is achieved under copper depletion, whereas normal copper concentrations in standard medium lead to low expression rates. We show here the application of this system for construction of a conditional knockout mutant for the ferrochelatase, which is an essential enzyme in heme biosynthesis. Using different amounts of copper in the medium we were able to control the amount of ferrochelatase in the cell resulting in a varying expression of the phenotype.

Light induced transmembrane proton gradient in artificial lipid vesicles reconstituted with photosynthetic reaction centers

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c₂ and K₄[Fe(CN)₆]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

Electron transfer between periplasmic formate dehydrogenase and cytochromes c in Desulfovibrio desulfuricans ATCC 27774

Desulfovibrio spp. are sulfate-reducing organisms characterized by having multiple periplasmic hydrogenases and formate dehydrogenases (FDHs). In contrast to enzymes in most bacteria, these enzymes do not reduce directly the quinone pool, but transfer electrons to soluble cytochromes c. Several studies have investigated electron transfer with hydrogenases, but comparatively less is known about FDHs. In this work we conducted experiments to assess potential electron transfer pathways resulting from formate oxidation in Desulfovibrio desulfuricans ATCC 27774. This organism can grow on sulfate and on nitrate, and contains a single soluble periplasmic FDH that includes a cytochrome c (3) like subunit (FdhABC(3)). It has also a unique cytochrome c composition, including two cytochromes c not yet isolated from other species, the split-Soret and nine-heme cytochromes, besides a tetraheme type I cytochrome c (3) (TpIc (3)). The FDH activity and cytochrome composition of cells grown with lactate or formate and nitrate or sulfate were determined, and the electron transfer between FDH and these cytochromes was investigated. We studied also the reduction of the Dsr complex and of the monoheme cytochrome c-553, previously proposed to be the physiological partner of FDH. FdhABC(3) was able to reduce the c-553, TpIc (3), and split-Soret cytochromes with a high rate. For comparison, the same experiments were performed with the [NiFe] hydrogenase from the same organism. This study shows that FdhABC(3) can directly reduce the periplasmic cytochrome c network, feeding electrons into several alternative metabolic pathways, which explains the advantage of not having an associated membrane subunit.

Electrochemical and infrared spectroscopic analysis of the interaction of the CuA domain and cytochrome c552 from Thermus thermophilus

The hydrophobically guided complex formation between the CuA fragment from Thermus thermophilus ba3 terminal oxidase and its electron transfer substrate, cytochrome c552, was investigated electrochemically. In the presence of the purified CuA fragment, a clear downshift of the c552 redox potential from 171 to 111mV±10mV vs SHE′ was found. Interestingly, this potential change fully matches complex formation with this electron acceptor site in other oxidases guided by electrostatic or covalent interactions. Redox induced FTIR difference spectra revealed conformational changes associated with complex formation and indicated the involvement of heme propionates. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Effects of different time administration of propofol on cytochrome c in cytoplasm in rat hippocampal neurons with hypoxia-reoxygenation injury

Objective To investigate the effects of different time administration of propofol on cytochrome c (Cyt c) in the cytoplasm in rat hippocampal neurons with hypoxia-reoxygenation (H/R) injury.Methods Primary cultured hippocampal neurons were randomly divided into 5 groups ( n =5 each):control group (group C),model of H/R injury group (group M),and different time administration of propofol groups (group Ⅰ,Ⅱ,Ⅲ ).In groups M,Ⅰ,Ⅱ and Ⅲ,the neurons were exposed to 95％ N2 + 5％ CO2 for6 h followed by 12 h reoxygenation.In groups Ⅰ, Ⅱ,Ⅲ,propofol was added to the culture medium before hypoxia,immediately after reoxygenation and at 2 h of reoxygenation (T0-2) respectively,with the final concentration of 20 μmol/L.The cell apoptosis was observed at T1,2 and at 24 h of reoxygenation ( T3 ) and the concentration of Cyt c in the cytoplasm was detected at T1-3.Results Compared with group C,the concentration of Cyt c in the cytoplasm was significantly increased at T1-3 in group M and at T1,2 in groups Ⅰ,Ⅱ,Ⅲ (P ＜ 0.05).Compared with group M,the concentration of Cyt c in the cytoplasm was significantly decreased at T1-3 in group I and at T1,2 in groups Ⅱ and Ⅲ ( P ＜0.05).The concentration of Cyt c in the cytoplasm was significantly higher at T1,2 in groups Ⅱ and Ⅲ than in group Ⅰ,and at T2 in group Ⅲ than in group Ⅱ ( P ＜ 0.05).The neuronal apoptosis was significantly decreased in groups Ⅰ,Ⅱ and Ⅲ as compared with group M.Conclusion Different time administration of propofol can reduce the mitochondrial Cyt c release to the cytoplasm,inhibit apoptosis in hippocampal neurons,and reduce H/R injury in rats,with better effect when given before hypoxia. Key words: Propofol; Hippocampus; Cell hypoxia; Cytochromes c

An Unusual Subtilisin-like Serine Protease Is Essential for Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH), an αβγ heterotrimer present in the periplasm of several Gram-negative bacteria, catalyzes the oxidative deamination of various aliphatic amines such as n-butylamine for assimilation as carbon and energy sources. The γ subunit of mature QHNDH contains a protein-derived quinone cofactor, cysteine tryptophylquinone, and three intrapeptidyl thioether cross-links between Cys and Asp or Glu residues. In its cytoplasmic nascent form, the γ subunit has a 28-residue N-terminal leader peptide that is necessary for the production of active QHNDH but must be removed in the following maturation process. Here, we describe the role of a subtilisin-like serine protease encoded in the fifth ORF of the n-butylamine-utilizing operon of Paracoccus denitrificans (termed ORF5) in QHNDH biogenesis. ORF5 disruption caused bacterial cell growth inhibition in n-butylamine-containing medium and production of inactive QHNDH, in which the γ subunit retained the leader peptide. Supply of plasmid-encoded ORF5 restored the cell growth and production of active QHNDH, containing the correctly processed γ subunit. ORF5 expressed in Escherichia coli but not its catalytic triad mutant cleaved synthetic peptides surrogating for the γ subunit leader peptide, although extremely slowly. The cleaved leader peptide remained unstably bound to ORF5, most likely as an acyl enzyme intermediate attached to the active-site Ser residue. These results demonstrate that ORF5 is essential for QHNDH biogenesis, serving as a processing protease to cleave the γ subunit leader peptide nearly in a disposable manner.

Isolation and Characterization of Homodimeric Type-I Reaction Center Complex from Candidatus Chloracidobacterium thermophilum, an Aerobic Chlorophototroph

The recently discovered thermophilic acidobacterium Candidatus Chloracidobacterium thermophilum is the first aerobic chlorophototroph that has a type-I, homodimeric reaction center (RC). This organism and its type-I RCs were initially detected by the occurrence of pscA gene sequences, which encode the core subunit of the RC complex, in metagenomic sequence data derived from hot spring microbial mats. Here, we report the isolation and initial biochemical characterization of the type-I RC from Ca. C. thermophilum. After removal of chlorosomes, crude membranes were solubilized with 0.1% (w/v) n-dodecyl β-D-maltoside, and the RC complex was purified by ion-exchange chromatography. The RC complex comprised only two polypeptides: the reaction center core protein PscA and a 22-kDa carotenoid-binding protein denoted CbpC. The absorption spectrum showed a large, broad absorbance band centered at ∼483 nm from carotenoids as well as smaller Q(y) absorption bands at 672 and 812 nm from chlorophyll a and bacteriochlorophyll a, respectively. The light-induced difference spectra of whole cells, membranes, and the isolated RC showed maximal bleaching at 840 nm, which is attributed to the special pair and which we denote as P840. Making it unique among homodimeric type-I RCs, the isolated RC was photoactive in the presence of oxygen. Analyses by optical spectroscopy, chromatography, and mass spectrometry revealed that the RC complex contained 10.3 bacteriochlorophyll a(P), 6.4 chlorophyll a(PD), and 1.6 Zn-bacteriochlorophyll a(P)' molecules per P840 (12.8:8.0:2.0). The possible functions of the Zn-bacteriochlorophyll a(P)' molecules and the carotenoid-binding protein are discussed.

Photo-induced electron transfer in intact cells of Rubrivivax gelatinosus mutants deleted in the RC-bound tetraheme cytochrome: Insight into evolution of photosynthetic electron transport

Deletion of two of the major electron carriers, the reaction center-bound tetrahemic cytochrome and the HiPIP, involved in the light-induced cyclic electron transfer pathway of the purple photosynthetic bacterium, Rubrivivax gelatinosus, significantly impairs its anaerobic photosynthetic growth. Analysis on the light-induced absorption changes of the intact cells of the mutants shows, however, a relatively efficient photo-induced cyclic electron transfer. For the single mutant lacking the reaction center-bound cytochrome, we present evidence that the electron carrier connecting the reaction center and the cytochrome bc1 complex is the High Potential Iron–sulfur Protein. In the double mutant lacking both the reaction center-bound cytochrome and the High Potential Iron–sulfur Protein, this connection is achieved by the high potential cytochrome c8. Under anaerobic conditions, the halftime of re-reduction of the photo-oxidized primary donor by these electron donors is 3 to 4 times faster than the back reaction between P+ and the reduced primary quinone acceptor. This explains the photosynthetic growth of these two mutants. The results are discussed in terms of evolution of the type II RCs and their secondary electron donors.