Electron transfer in cyanobacterial photosystem I: II. Determination of forward electron transfer rates of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.     

Specific mutagenesis of the rieske iron-sulfur protein in Rhodobacter sphaeroides shows that both the thermodynamic gradient and the pK of the oxidized form determine the rate of quinol oxidation by the bc(1) complex

In the Rieske iron-sulfur protein (ISP) of the ubiquinol:cytochrome c(2) oxidoreductase (bc(1) complex) of Rhodobacter sphaeroides, residue Tyr 156 is located close to the iron-sulfur cluster. Previous studies of the equivalent residue in both Saccharomyces cerevisiae [Denke, E., Merbitz-Zahradnik, T., Hatzfeld, O. M., Snyder, C. H., Link, T. A., and Trumpower, B. L. (1998) J. Biol. Chem. 273, 9085-9093] and Paracoccus denitrificans [Schroter, T., Hatzfeld, O. M., Gemeinhardt, S., Korn, M., Friedrich, T., Ludwig, B. , and Link, T. A. (1998) Eur. J. Biochem. 255, 100-106] have indicated that mutations at this site can lead to modifications in the redox potential of the ISP. To study the effect of similar modifications on the thermodynamic behavior and kinetics of partial reactions of the bc(1) complex upon flash activation, we have constructed four mutant strains of Rb. sphaeroides where Tyr 156 was mutated to His, Leu, Phe, or Trp. The bc(1) complex was assembled and able to support photosynthetic growth in all mutants. Three substitutions (Leu, Phe, Trp) led to alteration of the midpoint potential (E(m)) of the ISP and a slowing in rate of quinol oxidation, suggesting that electron transfer from quinol to the oxidized ISP controls the overall rate and that this step includes the high activation barrier. The Trp mutation led to an increase of approximately 1 pH unit in the pK value of the oxidized ISP. The pH dependence of the rate of quinol oxidation in this mutant was also shifted up by approximately 1 pH unit, showing the importance of the protonation state of the ISP for quinol oxidation. This provides support for a model in which the dissociated form of the oxidized ISP is required for formation of the enzyme-substrate complex [Ugulava, N., and Crofts, A. R. (1998) FEBS Lett. 440, 409-413].     

Chromatophore heterogeneity explains phenomena seen in Rhodobacter sphaeroides previously attributed to supercomplexes

It is generally considered that metabolic reactions are well described by homogeneous kinetic models in which the reaction phase is statistically uniform. In membranes, especially in photosynthetic systems where the protein complement is high, it has recently been recognized that effects of local heterogeneity might contribute additional factors that perturb the kinetic behavior, and require more extensive treatment. We show in this paper that statistical heterogeneity in vesicular systems can also contribute to quite marked discrepancies from the behavior expected from standard kinetic and thermodynamic models, for reactions involving free diffusion in the aqueous phase. We explain the kinetic and thermodynamic effects observed in studies of photosynthetic electron transfer in cells and chromatophores from Rhodobacter sphaeroides previously attributed to supercomplexes, in terms of a model based on heterogeneity in distribution of electron transfer components among the chromatophore population. We discuss examples of data inconsistent with the supercomplex model, but well explained by the heterogeneity model.     

Mechanism of ubiquinol oxidation by the bc(1) complex: role of the iron sulfur protein and its mobility

Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and structures with inhibitors in place, show a 16-22 A displacement of the [2Fe-2S] cluster and the position of the C-terminal extrinsic domain of the iron sulfur protein. None of the structures shows a static configuration that would allow catalysis of all partial reactions of quinol oxidation. We have suggested that the different conformations reflect a movement of the subunit necessary for catalysis. The displacement from an interface with cytochrome c(1) in native crystals to an interface with cytochrome b is induced by stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and involves ligand formation between His-161 of the [2Fe-2S] binding cluster and the inhibitor. The movement is a rotational displacement, so that the same conserved docking surface on the iron sulfur protein interacts with cytochrome c(1) and with cytochrome b. The mobile extrinsic domain retains essentially the same tertiary structure, and the anchoring N-terminal tail remains in the same position. The movement occurs through an extension of a helical segment in the short linking span. We report details of the protein structure for the two main configurations in the chicken heart mitochondrial complex and discuss insights into mechanism provided by the structures and by mutant strains in which the docking at the cytochrome b interface is impaired. The movement of the iron sulfur protein represents a novel mechanism of electron transfer, in which a tethered mobile head allows electron transfer through a distance without the entropic loss from free diffusion.     

Electron transfer from plastocyanin to the photosystem I reaction center in mutants with increased potential of the primary donor in Chlamydomonas reinhardtii

The dependence of the P(700)(+)/P(700) midpoint potential on kinetics of reduction of P(700)(+) in vivo has been examined in a series of site-directed mutants of Chlamydomonas reinhardtii in which the histidyl axial ligand to the Mg(2+) of the P(700) chlorophyll a has been changed to several different amino acids. In wild-type photosystem I, the potential of P(700)(+)/P(700) is 447 mV and the in vivo half-time of P(700)(+) reduction by its natural donor, plastocyanin, is 4 micros. Substitution of the axial histidine ligand with cysteine increases the potential of P(700)(+)/P(700) to 583 mV and changes the rate of P(700)(+) reduction to 0.8 micros. Mutants with a range of potentials between 447 and 583 mV show a strong correlation of the P(700)(+)/P(700) potential to the rate of reduction of P(700)(+) by plastocyanin. There is also an increase in the rate of photosystem I-mediated electron transfer from the artificial electron donor DCPIP to methyl viologen in thylakoid membranes. The results indicate that the overall rate constant of P(700)(+) reduction is determined by the rate of electron transfer between the copper and P(700)(+) and confirmed that in vivo there is a preformed complex between plastocyanin and photosystem I. Using approximations of the Marcus electron transfer theory, it is possible to estimate that the distance between the copper of plastocyanin and P(700)(+) is approximately 15 A. On the basis of this distance, the plastocyanin docking site should lie in a 10 A hollow formed by the lumenal exposed loops between transmembrane helices i and j of PsaA and PsaB.     

Expression and One-Step Purification of a Fully Active Polyhistidine-Tagged Cytochromebc1Complex fromRhodobacter sphaeroides

ThefbcBandfbcCgenes encoding cytochromesbandc₁of thebc₁complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were madein vitroin a pUC-derived background using PCR amplification. The modifiedfbcoperons were transferred to a pRK derivative plasmid, and this was used to transform thefbc⁻strain ofRhodobacter sphaeroides,BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of thebc₁complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA–Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of hemec₁, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutantbc₁complex, for spectroscopy and structural studies.     

Biochemical and biophysical characterization of photosystem I from phytoene desaturase and zeta-carotene desaturase deletion mutants of Synechocystis Sp. PCC 6803: evidence for PsaA- and PsaB-side electron transport in cyanobacteria

In photosystem I, oxidation of reduced acceptor A(1)(-) through iron-sulfur cluster F(X) is biphasic with half-times of approximately 5-30 ns ("fast" phase) and approximately 150-300 ns ("slow" phase). Whether these biphasic kinetics reflect unidirectional electron transfer, involving only the PsaA-side phylloquinone or bi-directional electron transfer, involving both the PsaA- and PsaB-side phylloquinones, has been the source of some controversy. Brettel (Brettel, K. (1988) FEBS Lett. 239, 93-98) and Joliot and Joliot (Joliot, P., and Joliot, A. (1999) Biochemistry 38, 11130-11136) have attributed to nearby carotenoids electrochromic band shifts, accompanying A(1) reduction, centered at approximately 450 and 500-510 nm. As a test of these assignments, we separately deleted in Synechocystis sp. PCC 6803 the genes that encode phytoene desaturase (encoded by crtP (pds)) and zeta-carotene desaturase (encoded by crtQ (zds)). The pds(-) and zds(-) strains synthesize phytoene and zeta-carotene, respectively, both of which absorb to shorter wavelength than beta-carotene. Compared with wild type, the mutant A(1)(-) (FeS) - A(1)(FeS)(-) difference spectra, measured in cells and photosystem I complexes, retain the electrochromic band shift centered at 450 nm but show a complete loss of the electrochromic band shifts centered at 500-510 nm. Thus, the latter clearly arise from beta-carotene. In the wild type, the electrochromic band shift of the slow phase (centered at 500 nm) is shifted by 6 nm to the blue compared with the fast phase (centered at 506 nm). Thus, the carotenoid pigments acting as electrochromic markers during the fast and slow phases of A(1)(-) oxidation are different, indicating the involvement of both the PsaA- and the PsaB-side phylloquinones in photosystem I electron transport.     

Expression and one-step purification of a fully active polyhistidine-tagged cytochrome bc1 complex from Rhodobacter sphaeroides

The fbcB and fbcC genes encoding cytochromes b and c1 of the bc1 complex were extended with a segment to encode a polyhistidine tag linked to their C-terminal sequence allowing a one-step affinity purification of the complex. Constructions were made in vitro in a pUC-derived background using PCR amplification. The modified fbc operons were transferred to a pRK derivative plasmid, and this was used to transform the fbc- strain of Rhodobacter sphaeroides, BC17. The transformants showed normal rates of growth. Chromatophores prepared from these cells showed kinetics of turnover of the bc1 complex on flash activation which were essentially the same as those from wild-type strains, and analysis of the cytochrome complement and spectral and thermodynamic properties by redox potentiometry showed no marked difference from the wild type. Chromatophores were solubilized and mixed with Ni-NTA-Sepharose resin. A modification of the standard elution protocol in which histidine replaced imidazole increased the activity 20-fold. Imidazole modified the redox properties of heme c1, suggesting ligand displacement and inactivation when this reagent is used at high concentration. The purified enzyme contained all four subunits in an active dimeric complex. This construction provides a facile method for preparation of wild-type or mutant bc1 complex, for spectroscopy and structural studies.     

Discovery of lung cancer biomarkers by profiling the plasma proteome with monoclonal antibody libraries

A challenge in the treatment of lung cancer is the lack of early diagnostics. Here, we describe the application of monoclonal antibody proteomics for discovery of a panel of biomarkers for early detection (stage I) of non-small cell lung cancer (NSCLC). We produced large monoclonal antibody libraries directed against the natural form of protein antigens present in the plasma of NSCLC patients. Plasma biomarkers associated with the presence of lung cancer were detected via high throughput ELISA. Differential profiling of plasma proteomes of four clinical cohorts, totaling 301 patients with lung cancer and 235 healthy controls, identified 13 lung cancer-associated (p < 0.05) monoclonal antibodies. The monoclonal antibodies recognize five different cognate proteins identified using immunoprecipitation followed by mass spectrometry. Four of the five antigens were present in non-small cell lung cancer cells in situ. The approach is capable of generating independent antibodies against different epitopes of the same proteins, allowing fast translation to multiplexed sandwich assays. Based on these results, we have verified in two independent clinical collections a panel of five biomarkers for classifying patient disease status with a diagnostics performance of 77% sensitivity and 87% specificity. Combining CYFRA, an established cancer marker, with the panel resulted in a performance of 83% sensitivity at 95% specificity for stage I NSCLC.     

Kinetics and pathways of charge recombination in photosystem II

The mechanism of charge recombination of the S(2)Q(A)(-) state in photosystem II was investigated by modifying the free energy gap between the quinone acceptor Q(A) and the primary pheophytin acceptor Ph. This was done either by changing the midpoint potential of Ph (using mutants of the cyanobacterium Synechocystis with a modified hydrogen bond to this cofactor), or that of Q(A) (using different inhibitors of the Q(B) pocket). The results show that the recombination rate is dependent on the free energy gap between Ph and Q(A), which confirms that the indirect recombination pathway involving formation of Ph(-) has a significant contribution. In the mutant with the largest free energy gap, direct electron transfer from Q(A)(-) to P(+) predominates. The temperature dependence of the recombination rate was investigated, showing a lower activation enthalpy in this mutant compared with the WT. The data allow the determination of the rate of the direct route and of its relative weight in the various strains. The set of currently accepted values for the midpoint potentials of the Q(A)/Q(A)(-), Ph/Ph(-), and P(+)/P* couples is not consistent with the relatively rapid rate of the indirect recombination pathway found here, nor with the 3% yield of delayed fluorescence as previously estimated by de Grooth and van Gorkom (1981, Biochim. Biophys. Acta 635, 445-456). It is argued that a likely explanation is that the midpoint potentials of the two latter couples are more positive than believed due to electrostatic interactions. If such is the case, the estimation of the midpoint potential of the P(+)/P and S(2)/S(1) couples must also be revised upward, with values of 1260 and 1020 mV, respectively.