Photosynthetic water oxidation at high O2 backpressure monitored by delayed chlorophyll fluorescence

The atmospheric dioxygen is produced by photosynthetic organisms. This light-driven process culminates in what appears as one step: a four-electron abstraction from two water molecules bound to the Mn4Ca complex of photosystem II. Recently, an intermediate of the O2-producing reaction sequence was stabilized by elevated oxygen backpressure and detected by UV flash photometry [Clausen, J., and Junge, W. (2004) Nature 430, 480]. We scrutinized its properties by delayed chlorophyll fluorescence measurements. Half-suppression of oxygen evolution was observed at a similar O2 pressure of 2.3 bar, as previously, now with photosystem II membrane particles from spinach, without artificial electron acceptors, and at a high signal-to-noise ratio. The data are tentatively interpreted as the stabilization of a 2-fold oxidized state of the catalytic center (S2*) with bound peroxide and its slow conversion into the normal S2 state by the release of peroxide.     

Evidence against bicarbonate bound in the O2-evolving complex of photosystem II

The oxidation of water to molecular oxygen by photosystem II (PSII) is inhibited in bicarbonate-depleted media. One contribution to the inhibition is the binding of bicarbonate to the non-heme iron, which is required for efficient electron transfer on the electron-acceptor side of PSII. There are also proposals that bicarbonate is required for formation of O 2 by the manganese-containing O 2-evolving complex (OEC). Previous work indicates that a bicarbonate ion does not bind reversibly close to the OEC, but it remains possible that bicarbonate is bound sufficiently tightly to the OEC that it cannot readily exchange with bicarbonate in solution. In this study, we have used NH 2OH to destroy the OEC, which would release any tightly bound bicarbonate ions from the active site, and mass spectrometry to detect any released bicarbonate as CO 2. The amount of CO 2 per PSII released by the NH 2OH treatment is observed to be comparable to the background level, although N 2O, a product of the reaction of NH 2OH with the OEC, is detected in good yield. These results strongly argue against tightly bound bicarbonate ions in the OEC.     

Quantitative assessment of intrinsic carbonic anhydrase activity and the capacity for bicarbonate oxidation in photosystem II

On the basis of equilibrium isotopic distribution experiments using (18)O-labeled water, it is generally accepted that water is the sole substrate for O(2) production by photosystem II (PSII). Nevertheless, recent studies indicating a direct interaction between bicarbonate and the donor side of PSII have been used to hypothesize that bicarbonate may have been a physiologically important substrate for O(2) production during the evolution of PSII [Dismukes, G. C., Klimov, V. V., Baranov, S. V., Kozlov, Y. N., DasGupta, J., and Tyryshikin, A. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 2170-2175]. To test out this hypothesis and to determine whether contemporary oxygenic organisms have the capacity to oxidize bicarbonate, we employed special rapid-mixing isotopic experiments using (18)O/(13)C-labeled bicarbonate to quantify the inherent carbonic anhydrase activity in PSII samples and the potential flux of oxygen from bicarbonate into the photosynthetically produced O(2). The measurements were made on PSII samples prepared from spinach, Thermosynechococcus elongatus, and Arthrospira maxima. For the latter organism, a strain was used that grows naturally in an alkaline, high (bi)carbonate soda lake in Africa. The results reveal that bicarbonate is not the substrate for O(2) production in these contemporary oxygenic photoautotrophs when assayed under single turnover conditions.     

Feedforward non-Michaelis-Menten mechanism for CO(2) uptake by Rubisco: contribution of carbonic anhydrases and photorespiration to optimization of photosynthetic carbon assimilation

Rubisco, the most abundant protein serving as the primary engine generating organic biomass on Earth, is characterized by a low catalytic constant (in higher plants approx. 3s(-1)) and low specificity for CO(2) leading to photorespiration. We analyze here why this enzyme evolved as the main carbon fixation engine. The high concentration of Rubisco exceeding the concentration of its substrate CO(2) by 2-3 orders of magnitude makes application of Michaelis-Menten kinetics invalid and requires alternative kinetic approaches to describe photosynthetic CO(2) assimilation. Efficient operation of Rubisco is supported by a strong flux of CO(2) to the chloroplast stroma provided by fast equilibration of bicarbonate and CO(2) and forwarding the latter to Rubisco reaction centers. The main part of this feedforward mechanism is a thylakoidal carbonic anhydrase associated with photosystem II and pumping CO(2) from the thylakoid lumen in coordination with the rate of electron transport, water splitting and proton gradient across the thylakoid membrane. This steady flux of CO(2) limits photosynthesis at saturating CO(2) concentrations. At low ambient CO(2) and correspondingly limited capacity of the bicarbonate pool in the stroma, its depletion at the sites of Rubisco is relieved by utilizing O(2) instead of CO(2), i.e. by photorespiration, a process which supplies CO(2) back to Rubisco and buffers the redox state and energy level in the chloroplast. Thus, the regulation of Rubisco function aims to keep steady non-equilibrium levels of CO(2), NADPH/NADP and ATP/ADP in the chloroplast stroma and to optimize the condition of homeostatic photosynthetic flux of matter and energy.     

Simulations of the (1)H electron spin echo-electron nuclear double resonance and (2)H electron spin echo envelope modulation spectra of exchangeable hydrogen nuclei coupled to the S(2)-state photosystem II manganese cluster

The pulsed EPR methods of electron spin echo envelope modulation (ESEEM) and electron spin echo-electron nuclear double resonance (ESE-ENDOR) are used to investigate the proximity of exchangeable hydrogens around the paramagnetic S(2)-state Mn cluster of the photosystem II oxygen-evolving complex. Although ESEEM and ESE-ENDOR are both pulsed electron paramagnetic resonance techniques, the specific mechanisms by which nuclear spin transitions are observed are quite different. We are able to generate good simulations of both (1)H ESE-ENDOR and (2)H ESEEM signatures of exchangeable hydrogens at the S(2)-state cluster. The convergence of simulation parameters for both methods provides a high degree of confidence in the simulations. Several exchangeable protons-deuterons with strong dipolar couplings are observed. In the simulations, two of the close ( approximately 2.5 A) hydrogen nuclei exhibit strong isotropic couplings and are therefore most probably associated with direct substrate ligation to paramagnetic Mn. Another two of the close ( approximately 2.7 A) hydrogen nuclei show no isotropic couplings and are therefore most probably not contained in Mn ligands. We suggest that these proximal hydrogens may be associated with a Ca(2+)-bound substrate, as indicated in recent mechanistic proposals for O(2) formation.     

Flash-induced FTIR difference spectroscopy shows no evidence for the structural coupling of bicarbonate to the oxygen-evolving Mn cluster in photosystem II

Bicarbonate is known to be required for the maximum activity of photosystem II. Although it is well established that bicarbonate is bound to the nonheme iron to regulate the quinone reactions, the effect of bicarbonate on oxygen evolution is still controversial, and its binding site and exact physiological roles remain to be clarified. In this study, the structural coupling of bicarbonate to the oxygen-evolving center (OEC) was studied using Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra during the S-state cycle of OEC were recorded using the PSII core complexes from Thermosynechococcus elongatus in the presence of either unlabeled bicarbonate or (13)C-bicarbonate. The H (12)CO 3 (-)-minus-H (13)CO 3 (-) double difference spectra showed prominent bicarbonate bands at the first flash, whereas no appreciable bands were detected at the second to fourth flashes. The bicarbonate bands at the first flash were virtually identical to those from the nonheme iron, which was preoxidized by ferricyanide and photoreduced by a single flash, recorded using Mn-depleted PSII complexes. Using the bicarbonate bands of the nonheme iron as an internal standard, it was concluded that no bicarbonate band arising from OEC exists in the S-state FTIR spectra. This conclusion indicates that bicarbonate is not affected by the structural changes in OEC upon the four S-state transitions. It is thus strongly suggested that bicarbonate is neither a ligand to the Mn cluster nor a cofactor closely coupled to OEC, although the possibility cannot be fully excluded that nonexchangeable bicarbonate exists in OEC as a constituent of the Mn-cluster core. The data also provide strong evidence that bicarbonate does not function as a substrate or a catalytic intermediate. Bicarbonate may play major roles in the photoassembly process of the Mn cluster and in the stabilization of OEC by a rather indirect interaction.     

Effects of methanol on the S i -state transitions in photosynthetic water-splitting

From a chemical point of view methanol is one of the closest analogues of water. Consistent with this idea EPR spectroscopy studies have shown that methanol binds at-or at least very close to-the Mn(4)O(x)Ca cluster of photosystem II (PSII). In contrast, Clark-type oxygen rate measurements demonstrate that the O(2) evolving activity of PSII is surprisingly unaffected by methanol concentrations of up to 10%. Here we study for the first time in detail the effect of methanol on photosynthetic water-splitting by employing a Joliot-type bare platinum electrode. We demonstrate a linear dependence of the miss parameter for S( i ) state advancement on the methanol concentrations in the range of 0-10% (v/v). This finding is consistent with the idea that methanol binds in PSII with similar affinity as water to one or both substrate binding sites at the Mn(4)O(x)Ca cluster. The possibility is discussed that the two substrate water molecules bind at different stages of the cycle, one during the S(4) --> S(0) and the other during the S(2) --> S(3) transition.     

Oxygen ligand exchange at metal sites - implications for the O2 evolving mechanism of photosystem II

The mechanism for photosynthetic O2 evolution by photosystem II is currently a topic of intense debate. Important questions remain as to what is the nature of the binding sites for the substrate water and how does the O-O bond form. Recent measurements of the 18O exchange between the solvent water and the photogenerated O2 as a function of the S-state cycle have provided some surprising insights to these questions (W. Hillier, T. Wydrzynski, Biochemistry 39 (2000) 4399-4405). The results show that one substrate water molecule is bound at the beginning of the catalytic sequence, in the S0 state, while the second substrate water molecule binds in the S3 state or possibly earlier. It may be that the second substrate water molecule only enters the catalytic sequence following the formation of the S3 state. Most importantly, comparison of the observed exchange rates with oxygen ligand exchange in various metal complexes reveal that the two substrate water molecules are most likely bound to separate Mn(III) ions, which do not undergo metal-centered oxidations through to the S3 state. The implication of this analysis is that in the S1 state, all four Mn ions are in the +3 oxidation state. This minireview summarizes the arguments for this proposal.     

Cooperative binding of oxygen to the water-splitting enzyme in the filamentous cyanobacterium Oscillatoria chalybea

In the filamentous cyanobacterium Oscillatoria chalybea photolysis of water does not take place in the complete absence of oxygen. A catalytic oxygen partial pressure of 15x10(-6) Torr has to be present for effective water splitting to occur. By means of mass spectrometry we measured the photosynthetic oxygen evolution in the presence of H(2)(18)O in dependence on the oxygen partial pressure of the atmosphere and analysed the liberations of (16)O(2), (16)O(18)O and (18)O(2) simultaneously. The observed dependences of the light-induced oxygen evolution on bound oxygen yield sigmoidal curves. Hill coefficient values of 3.0, 3.1 and 3.2, respectively, suggest that the binding is cooperative and that four molecules of oxygen have to be bound per chain to the oxygen evolving complex. Oxygen seems to prime the water-splitting reaction by redox steering of the S-state system, putting it in the dark into the condition from which water splitting can start. It appears that in O. chalybea an interaction of oxygen with S(0) and S(1) leads to S(2) and S(3), thus yielding the typical oxygen evolution pattern in which even after extensive dark adaptation substantial amounts of Y(1) and Y(2) are found. The interacting oxygen is apparently reduced to hydrogen peroxide. Mass spectrometry permits to distinguish this highly specific oxygen requirement from the interaction of bulk atmospheric oxygen with the oxygen evolving complex of the cyanobacterium. This interaction leads to the formation H(2)O(2) which is decomposed under O(2) evolution in the light. The dependence on oxygen-partial pressure and temperature is analysed. Structural peculiarities of the cyanobacterial reaction centre of photosystem II referring to the extrinsic peptides might play a role.     

Reduction-induced inhibition and Mn(II) release from the photosystem II oxygen-evolving complex by hydroquinone or NH2OH are consistent with a Mn(III)/Mn(III)/Mn(IV)/Mn(IV) oxidation state for the dark-adapted enzyme

Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen-evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex. Specifically, the data support oxidation states of Mn(III)(2)/Mn(IV)(2) for the dark stable S(1) state of the O(2)-evolving complex. Activity and Mn(II) loss data were best modeled by assuming an S(1) --> S(-)(1) conversion of intermediate probability, a S(-)(1) --> S(-)(3) reaction of high probability, and subsequent step(s) of low probability. This model predicts that photosystem II Mn clusters that have undergone an initial reduction step become more reactive toward a second reduction, followed by a slower third reduction step. Analysis of the Mn(II) release parameters used to model the data suggests that the photosystem II manganese cluster consists of three Mn atoms that exhibit a facile reactivity with both reductants, and a single Mn that is reducible but sterically trapped at or near its binding site. Activity assays indicate that intact photosystem II centers reduced to S(-)(1) can evolve oxygen upon illumination, but that these centers are inactive in preparations depleted of the extrinsic 23 and 17 kDa polypeptides. Finally, it was found that a substantial population of the tyrosine D radical is reduced by hydroxylamine, but a smaller population reacts with hydroquinone over the course of a 24 h exposure to the reductant.     

Water-sensitive low-frequency vibrations of reaction intermediates during S-state cycling in photosynthetic water oxidation

In photosynthetic water oxidation, two water molecules are converted to an oxygen molecule through five reaction intermediates, designated S(n) (n = 0-4), at the catalytic Mn cluster of photosystem II. To understand the mechanism of water oxidation, changes in the chemical nature of the substrate water as well as the Mn cluster need to be defined during S-state cycling. Here, we report for the first time a complete set of Fourier transform infrared difference spectra during S-state cycling in the low-frequency (670-350 cm(-1)) region, in which interactions between the Mn cluster and its ligands can be detected directly, in PS II core particles from Thermosynechococcus elongatus. Furthermore, vibrations from oxygen and/or hydrogen derived from the substrate water and changes in them during S-state cycling were identified using multiplex isotope-labeled water, including H2(18)O, D2(16)O, and D2(18)O. Each water isotope affected the low-frequency S-state cycling spectra, characteristically. The bands sensitive only to (16)O/(18)O exchange were assigned to the modes from structures involving Mn and oxygen having no interactions with hydrogen, while the bands sensitive only to H/D exchange were assigned to modes from amino acid side chains and/or polypeptide backbones that associate with water hydrogen. The bands sensitive to both (16)O/(18)O and H/D exchanges were attributed to the structure involving Mn and oxygen structurally coupled with hydrogen in a direct or an indirect manner through hydrogen bonds. These bands include the changes of intermediate species derived from substrate water during the process of photosynthetic water oxidation.     

Monitoring water reactions during the S-state cycle of the photosynthetic water-oxidizing center: detection of the DOD bending vibrations by means of Fourier transform infrared spectroscopy

Photosynthetic water oxidation takes place in the water-oxidizing center (WOC) of photosystem II (PSII). To clarify the mechanism of water oxidation, detecting water molecules in the WOC and monitoring their reactions at the molecular level are essential. In this study, we have for the first time detected the DOD bending vibrations of functional D 2O molecules during the S-state cycle of the WOC by means of Fourier transform infrared (FTIR) difference spectroscopy. Flash-induced FTIR difference spectra upon S-state transitions were measured using the PSII core complexes from Thermosynechococcus elongatus moderately deuterated with D 2 (16)O and D 2 (18)O. D 2 (16)O-minus-D 2 (18)O double difference spectra at individual S-state transitions exhibited six to eight peaks arising from the D (16)OD/D (18)OD bending vibrations in the 1250-1150 cm (-1) region. This observation indicates that at least two water molecules, not in any deprotonated forms, participate in the reaction at each S-state transition throughout the cycle. Most of the peaks exhibited clear counter peaks with opposite signs at different transitions, reflecting a series of reactions of water molecules at the catalytic site. In contrast, negative bands at approximately 1240 cm (-1) in the S 2 --> S 3, S 3 --> S 0, and possibly S 0 --> S 1 transitions, for which no clear counter peaks were found in other transitions, can be interpreted as insertion of substrate water into the WOC from a water cluster in the proteins. The characteristics of the weakly D-bonded OD stretching bands were consistent with the insertion of substrate from internal water molecules in the S 2 --> S 3 and S 3 --> S 0 transitions. The results of this study show that FTIR detection of the DOD bending vibrations is a powerful method for investigating the molecular mechanism of photosynthetic water oxidation as well as other enzymatic reactions involving functional water molecules.     

Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.     

No evidence from FTIR difference spectroscopy that aspartate-170 of the D1 polypeptide ligates a manganese ion that undergoes oxidation during the S0 to S1, S1 to S2, or S2 to S3 transitions in photosystem II

On the basis of mutagenesis and X-ray crystallographic studies, Asp170 of the D1 polypeptide is widely believed to ligate the (Mn)4 cluster that is located at the catalytic site of water oxidation in photosystem II. Recent proposals for the mechanism of water oxidation postulate that D1-Asp170 ligates a Mn ion that undergoes oxidation during one or more of the S0 --> S1, S1 --> S2, and S2 --> S3 transitions. To test these hypotheses, we have compared the FTIR difference spectra of the individual S state transitions in wild-type* PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 with those in D1-D170H mutant PSII particles. Remarkably, our data show that the D1-D170H mutation does not significantly alter the mid-frequency regions (1800-1000 cm(-1)) of any of the FTIR difference spectra. Therefore, we conclude that the oxidation of the (Mn)4 cluster does not alter the frequencies of the carboxylate stretching modes of D1-Asp170 during the S0 --> S1, S1 --> S2, or S2 --> S3 transitions. The simplest explanation for these data is that the Mn ion that is ligated by D1-Asp170 does not increase its charge or oxidation state during any of these S state transitions. These data have profound implications for the mechanism of water oxidation. Either (1) the oxidation of the Mn ion that is ligated by D1-Asp170 occurs only during the transitory S3 --> S4 transition and serves as the critical step in the ultimate formation of the O-O bond or (2) the oxidation increments and O2 formation chemistry that occur during the catalytic cycle involve only the remaining Mn3Ca portion of the Mn4Ca cluster. Our data also show that, if the increased positive charge on the (Mn)4 cluster that is produced during the S1 --> S2 transition is delocalized over the (Mn)4 cluster, it is not delocalized onto the Mn ion that is ligated by D1-Asp170.     

The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate

We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.     

Calcium controls the assembly of the photosynthetic water-oxidizing complex: a cadmium(II) inorganic mutant of the Mn4Ca core

Perturbation of the catalytic inorganic core (Mn4Ca1OxCly) of the photosystem II-water-oxidizing complex (PSII-WOC) isolated from spinach is examined by substitution of Ca2+ with cadmium(II) during core assembly. Cd2+ inhibits the yield of reconstitution of O2-evolution activity, called photoactivation, starting from the free inorganic cofactors and the cofactor-depleted apo-WOC-PSII complex. Ca2+ affinity increases following photooxidation of the first Mn2+ to Mn3+ bound to the 'high-affinity' site. Ca2+ binding occurs in the dark and is the slowest overall step of photoactivation (IM1-->IM1* step). Cd2+ competitively blocks the binding of Ca2+ to its functional site with 10- to 30-fold higher affinity, but does not influence the binding of Mn2+ to its high-affinity site. By contrast, even 10-fold higher concentrations of Cd2+ have no effect on O2-evolution activity in intact PSII-WOC. Paradoxically, Cd2+ both inhibits photoactivation yield, while accelerating the rate of photoassembly of active centres 10-fold relative to Ca2+. Cd2+ increases the kinetic stability of the photooxidized Mn3+ assembly intermediate(s) by twofold (mean lifetime for dark decay). The rate data provide evidence that Cd2+ binding following photooxidation of the first Mn3+, IM1-->IM1*, causes three outcomes: (i) a longer intermediate lifetime that slows IM1 decay to IM0 by charge recombination, (ii) 10-fold higher probability of attaining the degrees of freedom (either or both cofactor and protein d.f.) needed to bind and photooxidize the remaining 3 Mn2+ that form the functional cluster, and (iii) increased lability of Cd2+ following Mn4 cluster assembly results in (re)exchange of Cd2+ by Ca2+ which restores active O2-evolving centres. Prior EPR spectroscopic data provide evidence for an oxo-bridged assembly intermediate, Mn3+(mu-O2(-))Ca2+, for IM1*. We postulate an analogous inhibited intermediate with Cd2+ replacing Ca2+.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.     

Inorganic carbon acquisition in two green marine Stichococcus species

The mechanism of inorganic carbon (C(i)) uptake was examined in the marine green microalgae Stichococcus cylindricus and Stichococcus minor. External carbonic anhydrase (CA) activity was not detected in either species, by potentiometric assay or by mass spectrometry. Photosynthetic characteristics of C(i) uptake indicate that both species have high apparent affinity for CO(2) with a low K(1/2) (CO(2)) of about 10 μm. The O(2) evolution rates in light exceeded the spontaneous CO(2) formation rate by 2.5-fold in both species, which thus have active bicarbonate uptake. Mass spectrometric monitoring of CO(2) and O(2) fluxes showed that rates of O(2) evolution exceeded those of CO(2) depletion by about three- and twofold in S. minor and S. cylindricus, respectively, and also showed, in cells photosynthesizing at pH 8.2, a rapid depletion of CO(2) upon illumination to a CO(2) compensation concentration of 15.42 and 12.03 μm in S. minor and S. cylindricus, respectively. Both species also exhibit active CO(2) uptake: addition of bovine CA at CO(2) compensation concentration caused a rapid rise in CO(2) as the CO(2) -HCO(3) (-) equilibrium was restored. Accumulation of unfixed C(i) by cells at pH 8.2 was calculated to be 84.33 mm in S. cylindricus, and 30.37 mm in S. minor to give internal accumulations of 23- and 8-fold, respectively, compared to the external C(i) concentration.     

Evidence for recycling of inorganic phosphate by wheat chloroplasts during photosynthesis at air levels of CO2 and O2

Phosphate recycling under photorespiratory conditions was investigated using intact wheat chloroplasts from Triticum aestivum (cv. Maris dove). A decline in the optimal Pi level needed to support steady-state photosynthesis was observed (a) as the bicarbonate supply became limiting, or (b) as oxygen concentrations were increased. Further, at subsaturating CO2 and elevated O2 (52%), photosynthetic induction periods were shortest in the absence of exogenous Pi, and severely extended by its addition. Thus, photosynthesis under low CO2 levels which favor ribulose 1,5 bisphosphate (RuBP) oxygenase activity and glycolate synthesis by chloroplasts decreases their dependency on exogenous Pi from the initial illumination of chloroplasts through to the attainment of steady state rates of O2 evolution. Uptake of phosphate (Pi) was directly measured at ambient O2 concentrations and showed the stoichiometry of O2 evolved to Pi consumed at 10 mmol/L bicarbonate (saturating) had a mean value of 3.0, and was increased to 5.4 at 2.5 mmol/L bicarbonate and to > 8.0 at 1.0 mmol/L bicarbonate. The observation is consistent with enhanced stromal recycling of Pi released during hydrolysis of phosphoglycolate produced in greater quantities as the ratio of RuBP carboxylase relative to oxygenase activities (vc/vo) declines. The theoretical relationship between vc/vo and O2/Pi stoichiometries was derived and compared favorably to experimental data obtained.     

The affinities for the two substrate water binding sites in the O(2) evolving complex of photosystem II vary independently during S-state turnover

The first determinations of substrate water binding to the O(2) evolving complex in photosystem II as a complete function of the S states have been made. H(2)(18)O was rapidly injected into spinach thylakoid samples preset in either the S(0), S(1), S(2), or S(3) states, and the rate of (18)O incorporation into the O(2) produced was determined by time-resolved mass spectrometry. For measurements at m/e = 34 (i.e., for the (16)O(18)O product), the rate of (18)O incorporation in all S states shows biphasic kinetics, reflecting the binding of the two substrate water molecules to the catalytic site. The slow phase kinetics yield rate constants at 10 degrees C of 8 +/- 2, 0.021 +/- 0.002, 2.2 +/- 0.3, and 1.9 +/- 0.2 s(-1) for the S(0), S(1), S(2), and S(3) states, respectively, while the fast phase kinetics yield a rate constant of 36.8 +/- 1.9 s(-1) for the S(3) state but remain unresolvable (>100 (s-1)) for the S(0), S(1), and S(2) states. Comparisons of the (18)O exchange rates reveal that the binding affinity for one of the substrate water molecules first increases during the S(0) to S(1) transition, then decreases during the S(1) to S(2) transition, but stays the same during the S(2) to S(3) transition, while the binding affinity for the second substrate water molecule undergoes at least a 5-fold increase on the S(2) to S(3) transition. These findings are discussed in terms of two independent Mn(III) substrate binding sites within the O(2) evolving complex which are separate from the component that accumulates the oxidizing equivalents. One of the Mn(III) sites may only first bind a substrate water molecule during the S(2) to S(3) transition.