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Govindjee

Publications and source records attributed to Govindjee.

At least 37 records · Page 2Linked to original sources
PubMed1992

Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g., Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+Q-A or P+Q-B, respectively; (b) the kinetics of electron transfer from Q-A to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (tau = 3.5 ps), followed by reduction of the bacteriopheophytin (tau = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (tau = 410 +/- 30 and 47 +/- 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type).(ABSTRACT TRUNCATED AT 400 WORDS)

Benzoquinones↗
PubMed1991

Arginine residues in the D2 polypeptide may stabilize bicarbonate binding in photosystem II of Synechocystis sp. PCC.

Bicarbonate (HCO3-) causes a significant and reversible stimulation of anion-inhibited electron flow in photosystem II of higher plants and cyanobacteria. To test if selected arginine (Arg) residues are involved in the binding of HCO3-, we utilized oligonucleotide-directed mutagenesis to construct Synechocystis sp. PCC 6803 mutants carrying mutations in Arg residues in the D2 protein. Measurements of oxygen evolution showed that the D2 mutants R233Q (arginine-233----glutamine) and R251S (arginine-251----serine) were 10-fold more sensitive to formate than the wild type. The formate concentration giving half-maximal inhibition of the steady-state oxygen evolution rate was 48 mM, 4.5 mM and 4 mM for the wild type, R233Q and R251S, respectively. Measurements of oxygen evolution in single-turnover flashes confirm that the mutants are more sensitive to formate than the wild type. Measurements of chlorophyll a fluorescence decay kinetics after the second saturating actinic flash indicated that, after formate treatment, the halftime of QA- oxidation was decreased by approximately a factor of 2, 4 and 6 in the wild type, R251S and R233Q, respectively. The recombination rate between QA- and S2 was approx. 2-fold slower in R251S and R233Q than in the wild type. In the presence of 100 mM sodium formate, reactivation of the Hill reaction by bicarbonate showed that the wild type had an apparent Km for bicarbonate of 0.5 mM, while the Km values for R233Q and R251S were 1.4 and 1.5 mM, respectively. We suggest that Arg-233 and Arg-251 in the D2 polypeptide contribute to stabilization of HCO3- binding in Photosystem II.

Arginine↗
PubMed1990

Differential sensitivity of bicarbonate-reversible formate effects on herbicide-resistant mutants of Synechocystis 6714.

Herbicide-resistant mutants of the cyanobacterium Synechocystis 6714, that are altered in specific amino acids in their D-1 protein, shows differential sensitivity to formate treatment. Measurements on oxygen yield in a sequence of flashes, chlorophyll (Chl) a fluorescence transients and Chl a fluorescence yield decay after a flash reveal that the resistance of cells to formate treatment is in the following (highest to lowest) order: [double mutant] A251V/F211S (Az V) greater than [single mutant] F211S (Az I) congruent to wild type greater than [single mutant] S264A (DCMU II-A). Significance of these results in terms of overlapping between the herbicide and bicarbonate binding niches on the D-1 protein is discussed.

Bicarbonates↗
PubMed1990

Chlorophyll a fluorescence lifetime distributions in open and closed photosystem II reaction center preparations.

We have measured the decay of chlorophyll a fluorescence at 4 degrees C under anaerobic conditions in stabilized photosystem II reaction center complex isolated from spinach, using multifrequency (2-400 MHz) cross-correlation phase fluorometry. Examination of our data shows that although the fluorescence decay of open reaction centers (i.e., when both the electron donor P-680 and the electron acceptor pheophytin are capable of engaging in charge separation) can be analyzed as a multiexponential decay, another representation of the data is obtained when the decay is analyzed using a continuous distribution of lifetimes. Our results on the open reaction center differ from the two lifetime components of 25 ps and 35 ns published by Mimuro et al. (Biochim. Biophys. Acta 933 (1988) 478-486) for the D1-D2-cytochrome b-559 complex, obtained for F682 at 4 degrees C by a time-resolved photon-counting spectrofluorometer. When the reaction centers are closed by pretreatment with sodium dithionite and methyl viologen followed by exposure to laser excitation, conditions known to result in accumulation of reduced pheophytin, a dramatic decrease in the contribution of the slow lifetime component(s) is observed. These results suggest that the slow distribution lifetime component(s) in the 5-20 ns range originate(s) in the back reaction of the charge separated state. On the other hand, the fast lifetime component(s) in the picosecond range may be only partially related to the charge separation, since no dramatic change is observed upon closure of the reaction center. Perhaps, this component is related, in part, to the excitation energy migration among the various chromophores in the reaction center preparations.

Chlorophyll↗
PubMed1990

Chlorophyll a fluorescence transient as an indicator of active and inactive Photosystem II in thylakoid membranes.

Upon illumination, a dark-adapted photosynthetic sample shows time-dependent changes in chlorophyll (Chl) a fluorescence yield, known as the Kautsky phenomenon or the OIDPS transient. Based on the differential effects of electron acceptors such as 2,5-dimethyl-p-benzoquinone (DMQ) and 2,6-dichloro-p-benzoquinone (DCBQ) on Chl a fluorescence transients of spinach thylakoids, we suggest that the OID phase reflects the reduction of the electron acceptor QA to QA- in the inactive PS II (see Graan, T. and Ort, D. (1986) Diochim. Biophys. Acta 852, 320-330). In spinach thylakoids, heat-induced increase of the Chl a fluorescence yield is also differentially sensitive to the addition of DMQ and DCBQ suggesting that this increase is mainly on the 'I' level, and thus heating is suggested to convert active PS II to inactive PS II centers. The kinetics of decay of QA-, calculated from variable Chl a fluorescence, was analyzed into three exponential components (365-395 microseconds; 6-7 ms; and 1.4-1.7 s). In heated samples, the decay rate of variable Chl a fluorescence is slower than the normal back-reaction rate; there is a preponderance of the slow component that may be due, partly, to the active centers undergoing slow back reaction between QA- and the S2 state of the oxygen-evolving complex.

Benzoquinones↗
PubMed1990

Chlorophyll a fluorescence measurements of isolated spinach thylakoids obtained by using single-laser-based flow cytometry.

Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that chlorophyll a fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax-Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: 1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p-benzoquinone, phenyl-benzoquinone, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. 2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70 degrees C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. 3) Addition of an inhibitor diuron up to 100 microM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.

Chlorophyll↗
PubMed1989

Absence of a bicarbonate-depletion effect in electron transfer between quinones in chromatophores and reaction centers of Rhodobacter sphaeroides.

Higher plants, algae, and cyanobacteria are known to require bicarbonate ions for electron flow from the first stable electron acceptor quinone QA to the second electron acceptor quinone QB, and to the intersystem quinone pool. It has been suggested that in Photosystem II of oxygenic photosynthesis, bicarbonate ion functions to maintain the reaction center in a proper conformation and, perhaps, to provide the protons needed to stabilize the semiquinone (QB-). In this paper, we show that bicarbonate ions do not influence the electron flow, from the quinone QA to QB and beyond, in the photosynthetic bacterium Rhodobacter sphaeroides. No measurable effect of bicarbonate depletion, obtained by competition with formate, was observed on cytochrome b-561 reduction in chromatophores; on the flash-dependent oscillation of semiquinone formation in reaction centers; on electron transfer from QA- to QB; or on either the fast or slow recovery of the oxidized primary donor (P+) which reflects the P+QA- ----PQA or the P+QB- ----PQB reaction. The lack of an observed effect in Rhodobacter sphaeroides in contrast to the effect seen in Photosystem II is suggested to be due to the amino-acid sequence differences between the reaction centers of the two systems.

Bacterial Chromatophores↗
PubMed1986

Bicarbonate, not CO2, is the species required for the stimulation of Photosystem II electron transport.

Evidence is presented that the bicarbonate ion (HCO3-), not CO2, H2CO3 or CO32-, is the species that stimulates electron transport in Photosystem II from spinach (Spinacia oleracea). Advantage was taken of the pH dependence of the ratio of HCO3- to CO2 at equilibrium in order to vary effectively the concentration of one species while holding the other constant. The Hill reaction was stimulated in direct proportion with the equilibrium HCO3- concentration, but it was independent of the equilibrium CO2 concentration. The other two carbonic species, H2CO3 and CO32-, are also shown to have no direct involvement. It is suggested that HCO3- is the species which binds to the effector site.

2,6-Dichloroindophenol↗
PubMed1981

On the active species in bicarbonate stimulation of Hill reaction in thylakoid membranes.

At 5 degree C addition of CO2 or HCO3- to CO (HCO3-)-depleted thylakoids (containing 100 mM formate) initiates, within 10 s, the activation of the Hill reaction in light. In contrast to HCO3- addition, where there is a lag of 6-8 s, the activation by CO2 addition is almost instantaneous. With CO2, prior addition of carbonic anhydrase produces a lag of about 6 s that approaches the lag observed by the HCO3- addition. These data suggest that CO2 is an active species involved in stimulating the Hill reaction. Binding of CO2 (HCO3-) to a component on the external side of the thylakoid membranes is supported by the present study.

Bicarbonates↗
PubMed1981

Evidence for a close spatial location of the binding sites for CO2 and for photosystem II inhibitors.

1. CO2-depletion of thylakoid membranes results in a decrease of binding affinity of the Photosystem II (PS II) inhibitor atrazine. The inhibitory efficiency of atrazine, expressed as I50-concentration (50% inhibition) of 2,6-dichlorophenolindophenol reduction, is the same in CO2-depleted as well as in control thylakoids. This shows that CO2-depletion results in a complete inactivation of a part of the total number of electron transport chains. 2. A major site of action of CO2, which had previously been located between the two electron acceptor quinone molecule B (or R) and Photosystem II inhibitor atrazine as suggested by the following observations: (a) CO2-depletion results in a shift of the binding constant (kappa b) of [14C]atrazine to thylakoid membranes indicating a decreased affinity of atrazine to membrane; (b) trypsin treatment, which is known to modify the Photosystem II complex at the level of B, strongly diminishes CO2 stimulation of electron transport reactions in CO2-depleted membranes; and (c) thylakoids from atrazine-resistant plants, which contain a Photosystem II complex modified at the inhibitor binding site, show an altered CO2-stimulation of electron flow. 3. CO2-depletion does not produce structural changes in enzyme complexes involved in Photosystem II function of thylakoid membranes, as shown by freeze-fracture studies using electron microscopy.

Atrazine↗
PubMed1980

Effects of bulk pH and of monovalent and divalent cations on chlorophyll a fluorescence and electron transport in pea thylakoids.

Millimolar concentrations of monovalent cations enhance and divalent cations impede the redistribution (spill-over) of electronic excitation energy from Photosystem (PS) II to PS I in cation-depleted (sucrose-washed) thylakoids; this concept is based on chlorophyll a fluorescence and electron transport measurements over a narrow pH range around 7. We have tested the above concept in pea thylakoids over the pH range 5 to 9 by parallel measurements of various chlorophyll a fluorescence parameters (spectra, transients, and lifetimes at 77 K and 293 K, and polarization at 293 K) and of the rates of partial reactions of PSI and II. Our results provide the following information. (1) Mg2+ enhancement of fluorescence is maximum between 680 and 690 nm and minimum between 710 and 720 nm. (2) The optimum conditions for the observation of the Mg2+-induced enhancement of fluorescence are: wavelength of emission, 685 nm; concentratin of Mg2+, 10 mM, and pH, approximately 7.5. (3) Mg2+ decreases the efficiency of excitation redistribution from PS II to PS I over the pH range 6 to 9. (4) The antagonistic effects between Na+ and Mg2+ hold simultaneously for both the fluorescence intensity and lifetime, at physiological temperatures, only within the pH range 6 to 8. (5) Mg2+ enhances the light-limited electron transport rate through PS II in the pH range 5.4 to 8.2 and decreases that through PS I at pH 7.1 and 8.2. The % increase in PS II is, however, about twice the % decrease in PS I.

Cations↗
PubMed1979

Conformation and activity of chloroplast coupling factor exposed to low chemical potential of water in cells.

(1) Photophosphorylation, Ca2+-ATPase and Mg2+-ATPase activities of isolated chloroplasts were inhibited 55--65% when the chemical potential of water was decreased by dehydrating leaves to water potentials (psi w) of --25 bars before isolation of the plastids. The inhibition could be reversed in vivo by rehydrating the leaves. (2) These losses in activity were reflected in coupling factor (CF1) isolated from the leaves, since CF1 from leaves with low psi w had less Ca2+-ATPase activity than control CF1 and did not recouple phosphorylation in CF1-deficient chloroplasts. In contrast, CF1 from leaves having high psi w only partially recoupled phosphorylation by CF1-deficient chloroplasts from leaves havig low psi w. This indicated that low psi w affected chloroplast membranes as well as CF1 itself. (3) Coupling factor from leaves having low psi w had the same number of subunits, and the same electrophoretic mobility, and could be obtained with the same yields as CF1 from control leaves. However, direct measurements of fluorescence polarization, ultraviolet absorption, and circular dichroism showed that CF1 from leaves having low psi w differed from control CF1. The CF1 from leaves having low psi w also had decreased ability to bind fluorescent nucleotides (epsilon-ATP and epsilon-ADP). (4) Exposure of isolated CF1 to low psi w in vitro by preincubation in sucrose-containing media inhibited the Ca2+-ATPase activity of the protein in subsequent assays without sucrose. Inclusion of 5 or 10 mM Mg2+ in the preincubation medium markedly inhibited Ca2+-ATPase activity. (5) These results show that CF1 undergoes changes in cells which alter its phosphorylating ability. Since low cell psi w changed the spectroscopic properties but not other protein properties of CF1, the changes were most likely caused by altered confurn, photophosphorylation. The inhibition of ATPase activity in CF1 in vitro at low psi w and high ion concentration mimicked the change in activity seen in vivo.

Adenosine Triphosphatases↗
PubMed1978

Nuclear magnetic relaxation by the manganese in aqueous suspensions of chloroplasts.

Proton and oxygen-17 NMR relaxation rate (T1-1 and T2-1) data are presented for aqueous suspensions of dark-adapted chloroplasts. It is concluded from the dependence of the proton relaxation rates (PRR) upon Mn concentration that T1-1 and T2-1 are determined largely by the loosely bound Mn present in the chloroplast membranes. The frequency and temperature dependences of PRR are characteristic of Mn(II). The effects of oxidants (e.g., ferricyanide) and reductants (e.g., tetraphenylboron) on the PRR indicate that only about one-third to one-fourth of the loosely bound Mn is present in the dark-adapted chloroplasts as Mn(II), the remainder being in a higher oxidation state(s), probably Mn(III). The frequency dependence of the PRR for the chloroplast suspensions was fitted by a simplified form of the Solomon-Bloembergen-Morgan equations, and the following parameters were obtained: tauS = (1.1 +/- 0.1) X 10(-8) S; tauM = (2.2 +/- 0.2) X 10(-8) S; and B = (0.9 +/- 0.09) X 10(19). The oxygen-17 T1 and T2 data for suspensions before and after treatment with a detergent are consistent with the location of the manganese in the interior of the thylakoids. An analysis of the relaxation rates shows that the average lifetime of a water molecule inside a thylakoid is greater than 1 ms.

Chloroplasts↗