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C F Yocum

Publications and source records attributed to C F Yocum.

At least 37 records · Page 2Linked to original sources
PubMed1991

Calcium retards NH2OH inhibition of O2 evolution activity by stabilization of Mn2+ binding to photosystem II.

Calcium is required for oxidation of water to molecular oxygen by photosystem II; the Ca2+ demand of the reaction increases upon removal of 23- and 17-kDa extrinsic polypeptides from detergent-derived preparations of the photosystem. Employing the manganese reductant NH2OH as a probe to examine the function of Ca2+ in photosystem II reveals that (1) Ca2+ slows the rate of NH2OH inhibition of O2 evolution activity, but only in photosystem II membranes depleted of extrinsic proteins, (2) other divalent cations (Sr2+, Cd2+) that compete for the Ca2+ site also slow NH2OH inhibition, (3) Ca2+ is noncompetitive with respect to NH2OH, (4) in order to slow inhibition, Ca2+ must be present prior to the initiation of NH2OH reduction of manganese, and (5) Ca2+ appears not to interfere with NH2OH reduction of manganese. We conclude that the ability of Ca2+ to slow the rate of NH2OH inhibition arises from the site in photosystem II where Ca2+ normally stimulates O2 evolution and that the mechanism of this phenomenon arises from the ability of Ca2+ or certain surrogate metals to stabilize the ligation environment of the manganese complex.

Calcium↗
PubMed1990

Characterization by electron microscopy of isolated particles and two-dimensional crystals of the CP47-D1-D2-cytochrome b-559 complex of photosystem II.

A photosystem II complex containing the reaction center proteins D1 and D2, a 47-kDa chlorophyll-binding protein (CP47), and cytochrome b-559 was isolated with high yield, purity, and homogeneity; small but well-ordered two-dimensional crystals were prepared from the particles. The crystals and the isolated particles were analyzed by electron microscopy using negatively stained specimens. The information of 20 different digitized crystals was combined by alignment programs based on correlation methods to obtain a final average. The calculated diffraction pattern, with spots up to a resolution of 2.5 nm, and the optical diffraction pattern of a single crystal indicate that the plane group is p22121 (also called p2gg) and that the unit cell is rectangular with parameters of 23.5 x 16.0 nm, containing four stain-excluding monomers (two face-up and two face-down). In projection, the monomers have an asymmetrical shape with a length of 10 nm, a maximal width of 7.5 nm, and a height of 6 nm; their molecular mass is 175 +/- 40 kDa.

Chlorophyll↗
PubMed1990

EPR characterization of the CP47-D1-D2-cytochrome b-559 complex of photosystem II.

A photosystem II complex consisting of a 47-kDa chlorophyll-binding protein (CP47), the reaction center proteins D1 and D2, and cytochrome b-559 was characterized. Trace amounts of plastoquinone were found, indicating that the primary acceptor quinone QA has been extracted during purification. However, in the presence of ferricyanide, an EPR signal with the characteristic line shape and g value of the tyrosine radicals associated with photosystem II could be photoaccumulated in the majority of the reaction centers; in the absence of ferricyanide, or under low-temperature illumination conditions, a 9.5-11-G wide signal with a Gaussian line shape was observed at g = 2.003. Neither signal is observed in D1-D2-b-559 complexes, indicating that retention of CP47 produces a more native, but quinone-depleted photosystem II reaction center. The tyrosine radical photogenerated at room temperature can be trapped at cryogenic temperatures; results are presented showing that this radical can arise from tyrosine YZ, from tyrosine YD, or from both species. Low-temperature EPR spectroscopy also revealed a pronounced split signal with contributions at g = 6.05 and g = 5.75, which is attributed to high-spin, non-heme Fe3+ with axial ligation symmetry which is probably the non-heme iron associated with the acceptor side of photosystem II.

Chlorophyll↗
PubMed1989

Isolation and characterization of the 47 kDa protein and the D1-D2-cytochrome b-559 complex.

The 47 kDa polypeptide and a protein complex consisting of the D1 (32 kDa), D2 (34 kDa) and cytochrome b-559 (9 kDa) species were isolated from a Tris-washed Photosystem II core complex solubilized with dodecylmaltoside in the presence of LiClO4. Although the 43 kDa chlorophyll-binding protein is readily dissociated from the Photosystem II complex under our conditions, two cycles of exposure to high concentrations of detergent and LiClO4 were required for complete removal of the 47 kDa chlorophyll-binding protein from the D1-D2-cytochrome b-559 complex. Spectroscopic characterization of these two species revealed that the 47 kDa protein binds chlorophyll a, whereas the D1-D2-cytochrome b-559 complex shows an enrichment in Pheo a and heme on a chlorophyll basis. A spin-polarized EPR triplet can be observed at liquid helium temperatures in the D1-D2-cytochrome b-559 complex, but no such triplet is observed in the purified 47 kDa species. The zero-field splitting parameters of the P-680+ triplet indicate that the triplet spin is localized onto one chlorophyll molecule. Resonance Raman spectroscopy showed that: (i) beta-carotene is bound to the reaction center in its all-trans conformation; (ii) all chlorophyll a molecules are five-coordinate; and (iii) the C-9 keto group of one of the chlorine pigments is hydrogen-bonded. Our results support the proposal that the D1-D2 complex binds the P-680+ and Pheo a species that are involved in the primary charge separation.

Chlorophyll↗
PubMed1989

Evidence for a ferredoxin-dependent choline monooxygenase from spinach chloroplast stroma.

Chenopods synthesize betaine in the chloroplast via a two-step oxidation of choline: choline --> betaine aldehyde --> betaine. Our previous experiments with intact chloroplasts, and in vivo(18)O(2) labeling studies, led us to propose that the first step is mediated by a monooxygenase which uses photosynthetically generated reducing power (C Lerma, AD Hanson, D Rhodes [1988] Plant Physiol 88: 695-702). Here, we report the detection of such an activity in vitro. In the presence of O(2) and reduced ferredoxin, the stromal fraction from spinach (Spinacia oleracea) chloroplasts converted choline to betaine aldehyde at rates similar to those in intact chloroplasts (20-50 nanomoles per hour per milligram protein). Incorporation of (18)O from (18)O(2) by the in vitro reaction was demonstrated by fast atom bombardment mass spectrometry. Ferredoxin could be reduced either with thylakoids in the light, or with NADPH plus ferredoxin-NADP reductase in darkness; NADPH alone could not substitute for ferredoxin. No choline-oxidizing activity was detected in the stromal fraction of pea (Pisum sativum L.), a species that does not accumulate betaine. The spinach choline-oxidizing enzyme was stimulated by 10 millimolar Mg(2+), had a pH optimum close to 8, and was insensitive to carbon monoxide. The specific activity was increased threefold in plants growing in 200 millimolar NaCl. Gel filtration experiments gave a molecular weight of 98 kilodaltons for the choline-oxidizing enzyme, and provided no evidence for other electron carriers which might mediate the reduction of the 98-kilodalton enzyme by ferredoxin.

Journal Article↗
PubMed1987

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport : Effect of DCMU and the NADPH/NADP Ratio.

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O(2) (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP(+) ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.

Journal Article↗
PubMed1986

Polypeptides of the oxygen-evolving photosystem II complex. Immunological detection and biogenesis.

Oxygen-evolving photosystem II complex was isolated from spinach chloroplasts. The individual polypeptides of the complex were isolated from sodium dodecyl sulfate (SDS)-polyacrylamide gels and antibodies were raised in rabbits against these polypeptides. After washing of the isolation complex by 0.8 M Tris to release the extrinsic proteins, a distinct diffused protein band was revealed at the position of 33 kDa in SDS gels containing 4 M urea. When this band was electroeluted from the gel and subsequently electrophoresed on SDS gels, three distinct protein bands became apparent. Antibodies raised against each one of these polypeptides cross-reacted with the other two polypeptides to varying degrees but not with the other subunits of the complex. The three polypeptides were denoted as "34," "33," and "32" kDa and the 33 being the herbicide-binding protein. Using the antibodies, the relative amounts of the photosystem II polypeptides were followed during greening of etiolated spinach seedlings. While all three extrinsic polypeptides were present in etiolated leaves at relatively high amounts, the other polypeptides could not be detected prior to an approximate 6-h illumination period. Further illumination induced the appearance of all of the rest of the subunits in a relatively similar rate. The oxygen evolution activity was developed parallel to the increase in the amounts of these polypeptides. Therefore, the assembly of the active photosystem II during greening is a two-step process in contrast with the photosystem I reaction center, which is assembled step by step, and the rest of the chloroplast protein complexes, which are assembled by a concerted mechanism.

Antibody Specificity↗
PubMed1986

Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. I. Electrophoretic characterization and reconstitution of photophosphorylation.

The coupling factor ATPase (F1) from photosynthetic membranes of the cyanobacterium Spirulina platensis was purified to homogeneity by a combination of ion-exchange chromatography and sucrose density gradient centrifugation. The ATPase activity of purified Spirulina F1 is latent but can be elicited by trypsin treatment, resulting in specific activities (CaATPase) of 27-37 mumol Pi min-1 mg protein-1. On denaturing sodium dodecyl sulfate-polyacrylamide gradient gels, Spirulina F1 is resolved into five subunits with molecular weights of 53,400, 51,600, 36,000, 21,100, and 14,700, similar to the molecular weights of the subunits of spinach chloroplast coupling factor (CF1). As determined by native polyacrylamide gradient gel electrophoresis, the molecular weight of the Spirulina F1 holoenzyme was estimated to be 320,000, somewhat smaller than the estimated molecular weight of spinach CF1 (392,000). Spirulina F1 was shown to be an active coupling factor by its ability to reconstitute phenazine methosulfate-dependent cyclic photophosphorylation in membrane vesicles which had been depleted of coupling factor content by 2 M NaBr treatment. We estimate the Spirulina F1 content of membrane vesicles to be 1 F1 per 830 chlorophylls or 0.12 mol F1 mol P700(-1), based on the specific ATPase activities of the membrane vesicles and the purified Spirulina F1, the molecular weight of F1, and the P700 content of the vesicles.

Centrifugation, Density Gradient↗
PubMed1986

Properties of the cyanobacterial coupling factor ATPase from Spirulina platensis. II. Activity of the purified and membrane-bound enzymes.

Cyanobacterial (Spirulina platensis) photosynthetic membranes and isolated F1 ATPase were characterized with respect to ATP activity. The following results indicate that the regulation of expression of ATPase activity in Spirulina platensis is similar to that found in chloroplasts: the ATPase activity of Spirulina membranes and isolated F1 ATPase is mostly latent, a characteristic of chloroplast ATPase activity; treatments that elicit ATPase activity in higher plant chloroplast thylakoids and isolated chloroplast coupling factor (CF1) greatly stimulate the activity of Spirulina membranes and F1, and the cation specificity of chloroplast ATPase activity, e. g., light-induced membrane activity that is magnesium dependent and trypsin-activated CF1 activity that is calcium dependent, is also observed in Spirulina. Thus, an 8- to 15-fold increase in specific activity (to 13-15 mumol Pi min-1 mg chl-1) is obtained when Spirulina membranes are treated with trypsin (CaATPase) or with methanol (MgATPase): a light-induced, dithiothreitol-dependent MgATPase activity is also found in the membranes. Purified Spirulina F1 is a CaATPase when activated with trypsin (endogenous activity increases from 4 to 27-37 mumol Pi min-1 mg protein-1) or with dithiothreitol (5.6 mumol Pi min-1 mg-1), but a MgATPase when assayed with methanol (18-20 mumol Pi min-1 mg-1). The effects of varying calcium and ATP concentrations on the kinetics of trypsin-induced CaATPase activity of Spirulina F1 were examined. When the calcium concentration is varied at constant ATP concentration, the velocity plot shows a marked sigmoidicity. By varying Ca-ATP metal-nucleotide complex concentration at constant concentrations of free calcium or ATP, it is shown that the sigmoidicity is due to the effect of free ATP, which changes the Hill constant to 1.6 from 1.0 observed when the free calcium concentration is kept constant at 5 mM. Therefore not only is ATP an inhibitor but it is also an allosteric effector of Spirulina F1 ATPase activity. At 5 mM free calcium, the Km for teh Ca-ATP metal-nucleotide complex is 0.42 mM.

Adenosine Triphosphate↗
PubMed1985

Heparin inhibition of ferredoxin-NADP reductase in chloroplast thylakoid membranes.

Heparin, an anionic polysaccharide, inhibited the ferredoxin-catalyzed reduction of NADP in spinach chloroplast thylakoid membranes. Under the same conditions of assay, heparin did not interfere markedly with photoreduction of methyl viologen, anthraquinone sulfonate, or ferredoxin. A kinetic analysis of the heparin-induced interference with NADP photoreduction showed partial competitive inhibition. Heparin also interfered with NADPH oxidation by membrane-bound ferredoxin-NADP reductase (with dichlorophenol-indophenol as the acceptor) by a mechanism that involves partial competitive inhibition. This reaction was sensitive to the presence of salts; increasing ionic strength increases the heparin Ki for inhibition of NADPH oxidation. These results show that heparin binds to ferredoxin-NADP reductase, and in doing so interferes with binding to the reductase by both ferredoxin and NADP(H). Since heparin is redox inactive and does not interfere with the photophosphorylation reaction, it is a useful inhibitor of thylakoid membrane reactions which require the catalytic activity of ferredoxin-NADP reductase.

Catalysis↗
PubMed1982

Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis.

Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO(2) or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p-phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP(+) is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O(2) to H(2)O(2). Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations.

Journal Article↗
PubMed1981

Factors influencing hydroxylamine inactivation of photosynthetic water oxidation.

The kinetics of Mn release during NH2OH inactivation of the water oxidizing reaction is largely insensitive to the S-state present during addition of NH2OH. This appears to reflect reduction by NH2OH of higher S-states to a common more reduced state (S0 or S-1) which alone is susceptible to NH2OH inactivation. Sequences of saturating flashes with dark intervals in the range 0.2--5 S-1 effectively prevent NH2OH inactivation and the associated liberation of manganese. This light-induced protection disappears rapidly when the dark interval is longer than about 5 S. Under continuous illumination, protection against NH2OH inactivation is maximally effective at intensities in the range 10(3)--10(4) erg . cm-2 . S-1. This behavior differs from that of NH2OH-induced Mn release, which is strongly inhibited at all intensities greater than 10(3) erg . cm-2 . S-1. This indicates that two distinct processes are responsible for inactivation of water oxidation at high and low intensities. Higher S-states appear to be immune to the reaction by which NH2OH liberates manganese, although the overall process of water oxidation is inactivated by NH2OH in the presence of intense light. The light-induced protection phenomenon is abolished by 50 microM DCMU, but not by high concentrations of carbonyl cyanide m-chlorophenylhydrazone, which accelerates inactivation reactions of the water-splitting enzyme, Y (an ADRY reagent). The latter compound accelerates both inactivation of water oxidation and manganese extraction in the dark.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1981

Stoichiometry, inhibitor sensitivity, and organization of manganese associated with photosynthetic oxygen evolution.

Chloroplast thylakoid membranes isolated in the presence of EDTA retain high rates of O(2) evolution (>/=340 mumol.h(-1).mg chlorophyll(-1)) but contain no Mn(2+) that is detectable by electron paramagnetic resonance (EPR) at room temperature. The total Mn(2+) content of these preparations is 4.6 per 400 chlorophylls; 0.6 Mn(2+) can be released by addition of Ca(2+), a treatment that does not affect O(2) evolution. The remaining Mn(2+) (4 per 400 chlorophylls) appears to be functionally associated with O(2) evolution activity. Inhibition by Tris, NH(2)OH, or heat will release a small fraction of Mn(2+) from these membranes ( approximately 25% with Tris, for example). Addition of Ca(2+) further enhances Mn(2+) release so that for Tris and for NH(2)OH, 2 and 3, respectively, Mn(2+) per 400 chlorophylls are extracted from the O(2)-evolving complex. Based on the microwave power-saturation properties of the EPR signal IIf, which arises from an intermediate electron carrier in the water splitting process, it appears that one of the four Mn(2+) associated with photosystem II is uniquely sensitive to Tris. A new model is proposed for the organization and inhibitor sensitivity of manganese in the O(2)-evolving complex.

Journal Article↗
PubMed1981

Photophosphorylation Associated with Photosystem II: IV. KINETIC ANALYSES OF PHOTOSYSTEM II CYCLIC PHOTOPHOSPHORYLATION ACTIVITY: EVIDENCE FOR TWO CYCLIC REACTIONS.

Photosystem II-dependent cyclic photophosphorylation activity produced by addition of p-phenylenediamines to KCN-Hg-NH(2)OH-inhibited chloroplasts is the product of two separate reactions when a proton/electron donor is the catalyst. The activity observed with an electron donor as catalyst consists of a single reaction. One of the cyclic reactions, evoked by low (</=40 micromolar) concentrations of a proton/electron donor is sensitive to dibromothymoquinone and to perturbation of membrane organization by sonication. The second reaction, requiring higher catalyst concentrations, is less sensitive to either dibromothymoquinone or membrane perturbation. These results indicate that at low concentrations, proton/electron or electron donor catalysts act to produce a photosystem II cyclic reaction which is dependent on membrane-bound electron carriers. High concentrations of proton/electron donors, on the other hand, can produce a phosphorylation reaction in which the catalyst itself is largely responsible for cyclic activity.

Journal Article↗