Light on the cell cycle of the non-photosynthetic bacterium Ramlibacter tataouinensis.

Ramlibacter tataouinensis TTB310, a non-photosynthetic betaproteobacterium isolated from a semi-arid region of southern Tunisia, forms both rods and cysts. Cysts are resistant to desiccation and divide when water and nutrients are available. Rods are motile and capable of dissemination. Due to the strong correlation between sunlight and desiccation, light is probably an important external signal for anticipating desiccating conditions. Six genes encoding potential light sensors were identified in strain TTB310. Two genes encode for bacteriophytochromes, while the four remaining genes encode for putative blue light receptors. We determined the spectral and photochemical properties of the two recombinant bacteriophytochromes RtBphP1 and RtBphP2. In both cases, they act as sensitive red light detectors. Cyst divisions and a complete cyst-rod-cyst cycle are the main processes in darkness, whereas rod divisions predominate in red or far-red light. Mutant phenotypes caused by the inactivation of genes encoding bacteriophytochromes or heme oxygenase clearly show that both bacteriophytochromes are involved in regulating the rod-rod division. This process could favor rapid rod divisions at sunrise, after dew formation but before the progressive onset of desiccation. Our study provides the first evidence of a light-based strategy evolved in a non-photosynthetic bacterium to exploit scarse water in a desert environment.

Control by Metals of Staphylopine Dehydrogenase Activity during Metallophore Biosynthesis.

Enzymatic regulations are central processes for the adaptation to changing environments. In the particular case of metallophore-dependent metal uptake, there is a need to quickly adjust the production of these metallophores to the metal level outside the cell, to avoid metal shortage or overload, as well as waste of metallophores. In Staphylococcus aureus, CntM catalyzes the last biosynthetic step in the production of staphylopine, a broad-spectrum metallophore, through the reductive condensation of a pathway intermediate (xNA) with pyruvate. Here, we describe the chemical synthesis of this intermediate, which was instrumental in the structural and functional characterization of CntM and confirmed its opine synthase properties. The three-dimensional structure of CntM was obtained in an "open" form, in the apo state or as a complex with substrate or product. The xNA substrate appears mainly stabilized by its imidazole ring through a π-π interaction with the side chain of Tyr240. Intriguingly, we found that metals exerted various and sometime antagonistic effects on the reaction catalyzed by CntM: zinc and copper are moderate activators at low concentration and then total inhibitors at higher concentration, whereas manganese is only an activator and cobalt and nickel are only inhibitors. We propose a model in which the relative affinity of a metal toward xNA and an inhibitory binding site on the enzyme controls activation, inhibition, or both as a function of metal concentration. This metal-dependent regulation of a metallophore-producing enzyme might also take place in vivo, which could contribute to the adjustment of metallophore production to the internal metal level.

Connectivity of the intracytoplasmic membrane of Rhodobacter sphaeroides: a functional approach.

The photosynthetic apparatus in the bacterium Rhodobacter sphaeroides is mostly present in intracytoplasmic membrane invaginations. It has long been debated whether these invaginations remain in topological continuity with the cytoplasmic membrane, or form isolated chromatophore vesicles. This issue is revisited here by functional approaches. The ionophore gramicidin was used as a probe of the relative size of the electro-osmotic units in isolated chromatophores, spheroplasts, or intact cells. The decay of the membrane potential was monitored from the electrochromic shift of carotenoids. The half-time of the decay induced by a single channel in intact cells was about 6 ms, thus three orders of magnitude slower than in isolated chromatophores. In spheroplasts obtained by lysis of the cell wall, the single channel decay was still slower (~23 ms) and the sensitivity toward the gramicidin concentration was enhanced 1,000-fold with respect to isolated chromatophores. These results indicate that the area of the functional membrane in cells or spheroplasts is about three orders of magnitude larger than that of isolated chromatophores. Intracytoplasmic vesicles, if present, could contribute to at most 10% of the photosynthetic apparatus in intact cells of Rba. sphaeroides. Similar conclusions were obtained from the effect of a ∆pH-induced diffusion potential in intact cells. This caused a large electrochromic response of carotenoids, of similar amplitude as the light-induced change, indicating that most of the system is sensitive to a pH change of the external medium. A single internal membrane and periplasmic space may offer significant advantages concerning renewal of the photosynthetic apparatus and reallocation of the components shared with other bioenergetic pathways.

Estimation of the driving force for dioxygen formation in photosynthesis.

Photosynthetic water oxidation to molecular oxygen is carried out by photosystem II (PSII) over a reaction cycle involving four photochemical steps that drive the oxygen-evolving complex through five redox states Si (i = 0,…, 4). For understanding the catalytic strategy of biological water oxidation it is important to elucidate the energetic landscape of PSII and in particular that of the final S4 → S0 transition. In this short-lived chemical step the four oxidizing equivalents accumulated in the preceding photochemical events are used up to form molecular oxygen, two protons are released and at least one substrate water molecule binds to the Mn4CaO5 cluster. In this study we probed the probability to form S4 from S0 and O2 by incubating YD-less PSII in the S0 state for 2­3 days in the presence of (18)O2 and H2(16)O. The absence of any measurable (16,18)O2 formation by water-exchange in the S4 state suggests that the S4 state is hardly ever populated. On the basis of a detailed analysis we determined that the equilibrium constant K of the S4 → S0 transition is larger than 1.0 × 10(7) so that this step is highly exergonic. We argue that this finding is consistent with current knowledge of the energetics of the S0 to S4 reactions, and that the high exergonicity is required for the kinetic efficiency of PSII.

Sulphur shuttling across a chaperone during molybdenum cofactor maturation.

Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.

Characterization of bacteriophytochromes from photosynthetic bacteria: histidine kinase signaling triggered by light and redox sensing.

Bacteria detect environmental changes, thanks to two-component signal-transduction systems, composed, in general, of a sensor coupled to a histidine kinase and a DNA binding response regulator. Anoxygenic photosynthetic bacteria like Rhodopseudomonas (Rps.) palustris, possess a highly versatile metabolism and can grow via photosynthesis using light energy or via respiration through oxygen consumption. For photosynthetic bacteria, detecting changes in light quality or quantity, or in oxygen concentration, is therefore of prime importance for adjusting their metabolism for optimal development. A central role is played by bacteriophytochromes for light detection and initiation of regulatory responses. The switch of these chromoproteins between two photointerconvertible forms is the first event in the light-regulated cascade. This chapter describes in vitro and in vivo methods that have been successfully used to investigate the bacteriophytochrome dependent light regulation pathways, in several strains of Rps. palustris and Bradyrhizobium. These approaches range from biochemical and biophysical methods to genetic techniques. Such multiple approaches are indispensable for understanding these complex light-regulated pathway. In a first step, bacteriophytochromes and associated response regulators are overexpressed in Escherichia coli and purified. The spectral and kinetic properties of the two photointerconvertible forms of the purified bacteriophytochromes are then determined by biophysical approaches. Original spectral and kinetic properties found in some of the bacteriophytochromes that we studied necessitated the development of new methods for computing the spectra of the pure forms and the photoconversion yields. In vitro biochemical approaches help to assess the histidine kinase activity of bacteriophytochromes depending on light conditions, the phosphotransfer to response regulators and their affinity to promoter DNA sequences. Finally, gene inactivation tests the importance of specific genes in photosynthesis regulation under particular light and oxygen tension growth conditions. The methods described in this chapter are not restricted to the study of the light-transduction pathways of Rps. palustris and Bradyrhizobium strains but are applicable to the understanding of any bacterial light-regulatory system.

Excitation transfer connectivity in different purple bacteria: a theoretical and experimental study.

Photosynthetic membranes accommodate densely packed light-harvesting complexes which absorb light and convey excitation to the reaction center (RC). The relationship between the fluorescence yield (phi) and the fraction (x) of closed RCs is informative about the probability for an excitation reaching a closed RC to be redirected to another RC. In this work, we have examined in this respect membranes from various bacteria and searched for a correlation with the arrangement of the light-harvesting complexes as known from atomic force or electron microscopies. A first part of the paper is devoted to a theoretical study analyzing the phi(x) relationship in various models: monomeric or dimeric RC-LH1 core complexes, with or without the peripheral LH2 complexes. We show that the simple "homogeneous" kinetic treatment used here agrees well with more detailed master equation calculations. We also discuss the agreement between information derived from the present technique and from singlet annihilation experiments. The experimental results show that the enhancement of the cross section of open RCs due to excitation transfer from closed units varies from 1.5 to 3 depending on species. The ratio of the core to core transfer rate (including the indirect pathway via LH2) to the rate of trapping in open units is in the range of 0.5 to 4. It is about 1 in Rhodobacter sphaeroides and does not increase significantly in mutants lacking LH2-despite the more numerous contacts between the dimeric core complexes expected in this case. The connectivity in this bacterium is due in good part to the fast transfer between the two partners of the dimeric (RC-LH1-PufX)(2) complex. The connectivity is however increased in the carotenoidless and LH2-less strain R26, which we ascribe to an anomalous LH1. A relatively high connectivity was found in Rhodospirillum photometricum, although not as high as predicted in the calculations of Fassioli et al. (2010). This illustrates a more general discrepancy between the measured efficiency of core to core excitation transfer and theoretical estimates. We argue that the limited core to core connectivity found in purple bacteria may reflect a trade-off between light-harvesting efficiency and the hindrance to quinone diffusion that would result from too tightly packed LH complexes.

Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions.

Assimilation of atmospheric CO2 by photosynthetic organisms such as plants, cyanobacteria and green algae, requires the production of ATP and NADPH in a ratio of 3:2. The oxygenic photosynthetic chain can function following two different modes: the linear electron flow which produces reducing power and ATP, and the cyclic electron flow which only produces ATP. Some regulation between the linear and cyclic flows is required for adjusting the stoichiometric production of high-energy bonds and reducing power. Here we explore, in the green alga Chlamydomonas reinhardtii, the onset of the cyclic electron flow during a continuous illumination under aerobic conditions. In mutants devoid of Rubisco or ATPase, where the reducing power cannot be used for carbon fixation, we observed a stimulation of the cyclic electron flow. The present data show that the cyclic electron flow can operate under aerobic conditions and support a simple competition model where the excess reducing power is recycled to match the demand for ATP.

Thermoluminescence: theory.

Thermoluminescence (TL) probes the emission of luminescence associated with the de-trapping of a radical pair as the temperature is increased. This technique has proved useful for characterizing the energetic arrangement of cofactors in photosynthetic reaction centers. In the original TL theory, stemming from solid-state physics, the radical pair recombination was considered to coincide with the light-emitting process. In photosynthetic systems, however, recombination takes place through various routes among which the radiative pathway generally represents a relatively minor leak, and the theoretical framework must be modified accordingly. The radiative route is the one with the largest activation energy and is thus (still) more disfavored at low temperature, so that during the heating process, the TL peak tends to lag behind the decay of the radical pair. A consequence is that the integrated luminescence emission increases with the heating rate. In this article, we examine how the characteristics of the TL emission depend on the redox potentials of the cofactors, showing good agreement between theory and experimental studies on Photosystem (PS) II mutants. We also analyze the effect on (thermo-) luminescence of the connectivity of the light-harvesting pigment antenna, and show that while this should affect significantly luminescence kinetics at room temperature, the effect on TL is expected to be small.

Organisation and function of the Phaeospirillum molischianum photosynthetic apparatus.

We have investigated the organisation of the photosynthetic apparatus in Phaeospirillum molischianum, using biochemical fractionation and functional kinetic measurements. We show that only a fraction of the ATP-synthase is present in the membrane regions which contain most of the photosynthetic apparatus and that, despite its complicated stacked structure, the intracytoplasmic membrane delimits a single connected space. We find that the diffusion time required for a quinol released by the reaction centre to reach a cytochrome bc1 complex is about 260 ms. On the other hand, the reduction of the cytochrome c chain by the cytochrome bc1 complex in the presence of a reduced quinone pool occurs with a time constant of about 5 ms. The overall turnover time of the cyclic electron transfer is about 25 ms in vivo under steady-state illumination. The sluggishness of the quinone shuttle appears to be compensated, at least in part, by the size of the quinone pool. Together, our results show that P. molischianum contains a photosynthetic system, with a very different organisation from that found in Rhodobacter sphaeroides, in which quinone/quinol diffusion between the RC and the cytochrome bc1 is likely to be the rate-limiting factor for cyclic electron transfer.

Conformational control of the Q(A) to Q(B) electron transfer in bacterial reaction centers: evidence for a frozen conformational landscape below -25 degrees C.

The competition between the P(+)Q(A)(-) --> PQ(A) charge recombination (P, bacteriochlorophyll pair acting as primary photochemical electron donor) and the electron transfer to the secondary quinone acceptor Q(A)(-)Q(B) --> Q(A)Q(B)(-) (Q(A) and Q(B), primary and secondary electron accepting quinones) was investigated in chromatophores of Rb. capsulatus, varying the temperature down to -65 degrees C. The analysis of the flash-induced pattern for the formation of P(+)Q(A)Q(B)(-) shows that the diminished yield, when lowering the temperature, is not due to a homogeneous slowing of the rate constant k(AB) of the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer but to a distribution of conformations that modulate the electron transfer rate over more than 3 orders of magnitude. This distribution appears "frozen", as no dynamic redistribution was observed over time ranges > 10 s (below -25 degrees C). The kinetic pattern was analyzed to estimate the shape of the distribution of k(AB), showing a bell-shaped band on the high rate side and a fraction of "blocked" reaction centers (RCs) with very slow k(AB). When the temperature is lowered, the high rate band moves to slower rate regions and the fraction of blocked RCs increases at the expense of the high rate band. The RCs that recombine from the P(+)Q(A)Q(B)(-) state appear temporarily converted to a state with rapid k(AB), indicating that the stabilized state described by Kleinfeld et al. (Biochemistry 1984, 23, 5780-5786) is still accessible at -60 degrees C.

CYP201A2, a cytochrome P450 from Rhodopseudomonas palustris, plays a key role in the biodegradation of tributyl phosphate.

Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in nuclear fuel processing and chemical industries. Rhodopseudomonas palustris, one of the most metabolically versatile photosynthetic bacteria, is shown here to degrade TBP efficiently under photosynthetic conditions. This study shows that this O(2)- and NADPH/FMNH(2)-dependent process was also catalyzed when TBP was incubated with membrane-associated proteins extracted from this strain. The effects of several regulators of cytochrome P450 activity on the TBP consumption suggest a key role for a cytochrome P450 in this process. Disruption of the rpa0241 gene encoding a putative cytochrome P450 led to a 60% decrease of the TBP catabolism, whereas reintroducing the gene in the mutant restored the wild-type phenotype. The rpa0241 gene was expressed and purified in Escherichia coli. Characterization by UV-visible spectroscopy of the purified recombinant membrane-bound protein (CYP201A2) encoded by the rpa0241 gene revealed typical spectral characteristics of cytochrome P450 with a large spin state change of the heme iron associated with binding of TBP (K (d) approximately 65 microM). It is proposed that CYP201A2 catalyzes the initial step of the biodegradation process of TBP.

The unusually strong hydrogen bond between the carbonyl of Q(A) and His M219 in the Rhodobacter sphaeroides reaction center is not essential for efficient electron transfer from Q(A)(-) to Q(B).

In native reaction centers (RCs) from photosynthetic purple bacteria the primary quinone (QA) and the secondary quinone (QB) are interconnected via a specific His-Fe-His bridge. In Rhodobacter sphaeroides RCs the C4=O carbonyl of QA forms a very strong hydrogen bond with the protonated Npi of His M219, and the Ntau of this residue is in turn coordinated to the non-heme iron atom. The second carbonyl of QA is engaged in a much weaker hydrogen bond with the backbone N-H of Ala M260. In previous work, a Trp side chain was introduced by site-directed mutagenesis at the M260 position in the RC of Rb. sphaeroides, resulting in a complex that is completely devoid of QA and therefore nonfunctional. A photochemically competent derivative of the AM260W mutant was isolated that contains a Cys side chain at the M260 position (denoted AM260(W-->C)). In the present work, the interactions between the carbonyl groups of QA and the protein in the AM260(W-->C) suppressor mutant have been characterized by light-induced FTIR difference spectroscopy of the photoreduction of QA. The QA-/QA difference spectrum demonstrates that the strong interaction between the C4=O carbonyl of QA and His M219 is lost in the mutant, and the coupled CO and CC modes of the QA- semiquinone are also strongly perturbed. In parallel, a band assigned to the perturbation of the C5-Ntau mode of His M219 upon QA- formation in the native RC is lacking in the spectrum of the mutant. Furthermore, a positive band between 2900 and 2400 cm-1 that is related to protons fluctuating within a network of highly polarizable hydrogen bonds in the native RC is reduced in amplitude in the mutant. On the other hand, the QB-/QB FTIR difference spectrum is essentially the same as for the native RC. The kinetics of electron transfer from QA- to QB were measured by the flash-induced absorption changes at 780 nm. Compared to native RCs the absorption transients are slowed by a factor of about 2 for both the slow phase (in the hundreds of microseconds range) and fast phase (microseconds to tens of microseconds range) in AM260(W-->C) RCs. We conclude that the unusually strong hydrogen bond between the carbonyl of QA and His M219 in the Rb. sphaeroides RC is not obligatory for efficient electron transfer from QA- to QB.

Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for alpha-keto acid binding.

BACKGROUND: The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H+ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds alpha-keto acids in vitro. RESULTS: We describe the high-resolution crystal structures of TakP in its unliganded form and as a complex with sodium-pyruvate. The results show a limited "Venus flytrap" conformational change induced by substrate binding. In the liganded structure, a cation (most probably a sodium ion) is present and plays a key role in the association of the pyruvate to the protein. The structure of the binding pocket gives a rationale for the relative affinities of various ligands that were tested from a fluorescence assay. The protein appears to be dimeric in solution and in the crystals, with a helix-swapping structure largely participating in the dimer formation. A 30 A-long water channel buried at the dimer interface connects the two ligand binding cavities of the dimer. CONCLUSION: The concerted recruitment by TakP of the substrate group with a cation could represent a first step in the coupled transport of both partners, providing the driving force for solute import. Furthermore, the unexpected dimeric structure of TakP suggests a molecular mechanism of solute uptake by the dimeric ESR via a channel that connects the binding sites of the two monomers.

Evidence for delocalized anticooperative flash induced proton binding as revealed by mutants at the M266His iron ligand in bacterial reaction centers.

Bacterial reaction centers (RCs) convert light energy into chemical free energy via the double reduction and protonation of the secondary quinone electron acceptor, QB, to the dihydroquinone QBH2. Two RC mutants (M266His --> Leu and M266His --> Ala) with a modified ligand of the non-heme iron have been studied by flash-induced absorbance change spectroscopy. No important changes were observed for the rate constants of the first and second electron transfers between the first quinone electron acceptor, QA, and QB. However, in the M266HL mutant a destabilization of approximately 40 meV of the free energy level of QA- was observed, at variance with the M266HA mutant. The superposition of the three-dimensional X-ray structures of the three proteins in the QA region provides no obvious explanation for the energy modification in the M266HL mutant. The shift of the midpoint redox potential of QA/QA- in M266HL caused accelerated recombination of the charges in the P+ QA- state of the RCs where the native QA was replaced by a low potential anthraquinone (AQA). As previously reported for the native RCs, in the M266HL we observed a biphasicity of the P+ AQA- --> P AQA charge recombination. Interestingly, both phases present a similar acceleration in the M266HL mutant with respect to the wild type. The pH dependencies of the proton uptake upon QA- and QB- formations are superimposable in both mutants but very different from those of native RCs. The data measured in mutants are similar to those that we previously obtained on strains modified at various sites of the cytoplasmic region. The similarity of the response to these different mutations is puzzling, and we propose that it arises from a collective behavior of multiple acidic residues resulting in strongly anticooperative proton binding. The unspecific disappearance of the high pH band of proton uptake observed in all these mutants appears as the natural consequence of removing any member of an interactive proton cluster. This long range interaction also accounts for the similar responses to mutations of the proton uptake pattern induced by either QA- or QB-. We surmise that the presence of an extended protonated water H-bond network providing protons to QB is responsible for these effects.

A singular bacteriophytochrome acquired by lateral gene transfer.

Bacteriophytochromes are phytochrome-like proteins that mediate photosensory responses in various bacteria according to their light environment. The genome of the photosynthetic and plant-symbiotic Bradyrhizobium sp. strain ORS278 revealed the presence of a genomic island acquired by lateral transfer harboring a bacteriophytochrome gene, BrBphP3.ORS278, and genes involved in the synthesis of phycocyanobilin and gas vesicles. The corresponding protein BrBphP3.ORS278 is phylogenetically distant from the other (bacterio)phytochromes described thus far and displays a series of unusual properties. It binds phycocyanobilin as a chromophore, a unique feature for a bacteriophytochrome. Moreover, its C-terminal region is short and displays no homology with any known functional domain. Its dark-adapted state absorbs maximally around 610 nm, an unusually short wavelength for (bacterio)phytochromes. This form is designated as Po for orange-absorbing form. Upon illumination, a photo-reversible switch occurs between the Po form and a red (670 nm)-absorbing form (Pr), which rapidly backreacts in the dark. Because of this instability, illumination results in a mixture of the Po and Pr states in proportions that depend on the intensity. These uncommon features suggest that BrBphP3.ORS278 could be fitted to measure light intensity rather than color.

Commentary on: "Photosynthesis and negative entropy production" by Jennings and coworkers.

This commentary argues against the view that photochemical energy conversion violates the second law of thermodynamics, as expressed in a recent paper [R.C. Jennings, E. Engelmann, F. Garlaschi, A.P. Casazza, G. Zucchelli. Photosynthesis and negative entropy production. Biochim. Biophys. Acta 1709 (2005) 251-255]. The basic principles of free energy conversion by a photo-electrochemical cell are outlined, emphasizing the fact that the potential depends on the relative population of the excited state and thus on the illumination intensity.

Kinetic performance and energy profile in a roller coaster electron transfer chain: a study of modified tetraheme-reaction center constructs.

In many electron-transfer proteins, the arrangement of cofactors implies a succession of uphill and downhill steps. The kinetic implications of such arrangements are examined in the present work, based on a study of chimeric photosynthetic reaction centers obtained by expressing the tetraheme subunit from Blastochloris viridis in another purple bacterium, Rubrivivax gelatinosus. Site-directed mutations of the environment of heme c559, which is the immediate electron donor to the primary donor P, induced modifications of this heme's midpoint potential over a range of 400 mV. This resulted in shifts of the apparent midpoint potentials of the neighboring carriers, yielding estimates of the interactions between redox centers. At both extremities of the explored range, the energy profile of the electron-transfer chain presented an additional uphill step, either downstream or upstream from c559. These modifications caused conspicuous changes of the electron-transfer rate across the tetraheme subunit, which became approximately 100-fold slower in the mutants where the midpoint potential of c559 was lowest. A theoretical analysis of the kinetics is presented, predicting a displacement of the rate-limiting step when lowering the potential of c559. A reasonable agreement with the data was obtained when combining this treatment with the rates predicted by electron transfer theory for the individual rate constants.

Spectral and redox characterization of the heme ci of the cytochrome b6f complex.

Absorption spectra of the purified cytochrome b(6)f complex from Chlamydomonas reinhardtii were monitored as a function of the redox potential. Four spectral and redox components were identified: in addition to heme f and the two b hemes, the fourth component must be the new heme c(i) (also denoted x) recently discovered in the crystallographic structures. This heme is covalently attached to the protein, but has no amino acid axial ligand. It is located in the plastoquinone-reducing site Q(i) in the immediate vicinity of a b heme. Each heme titrated as a one-electron Nernst curve, with midpoint potentials at pH 7.0 of -130 mV and -35 mV (hemes b), +100 mV (heme c(i)), and +355 mV (heme f). The reduced minus oxidized spectrum of heme c(i) consists of a broad absorption increase centered approximately 425 nm. Its potential has a dependence of -60 mV/pH unit, implying that the reduced form binds one proton in the pH 6-9 range. The Q(i) site inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide, a semiquinone analogue, induces a shift of this potential by about -225 mV. The spectrum of c(i) matches the absorption changes previously observed in vivo for an unknown redox center denoted "G." The data are discussed with respect to the effect of the membrane potential on the electron transfer equilibrium between G and heme b(H) found in earlier experiments.

Charge recombination and thermoluminescence in photosystem II.

In the recombination process of Photosystem II (S(2)Q(A)(-)-->S(1)Q(A)) the limiting step is the electron transfer from the reduced primary acceptor pheophytin Ph(-) to the oxidized primary donor P(+) and the rate depends on the equilibrium constant between states S(2)PPhQ(A)(-) and S(1)P(+)Ph(-)Q(A). Accordingly, mutations that affect the midpoint potential of Ph or of P result in a modified recombination rate. A strong correlation is observed between the effects on the recombination rate and on thermoluminescence (TL, the light emission from S(2)Q(A)(-) during a warming ramp): a slower recombination corresponds to a large enhancement and higher temperature of the TL peak. The current theory of TL does not account for these effects, because it is based on the assumption that the rate-limiting step coincides with the radiative process. When implementing the known fact that the radiative pathway represents a minor leak, the modified TL theory readily accounts qualitatively for the observed behavior. However, the peak temperature is still lower than predicted from the temperature-dependence of recombination. We argue that this reflects the heterogeneity of the recombination process combined with the enhanced sensitivity of TL to slower components. The recombination kinetics are accurately fitted as a sum of two exponentials and we show that this is not due to a progressive stabilization of the charge-separated state, but to a pre-existing conformational heterogeneity.