Dissipation of the Proton Electrochemical Potential in Intact Chloroplasts (II. The pH Gradient Monitored by Cytochrome f Reduction Kinetics)

The potency of various uncouplers for collapsing the light-induced pH gradient across thylakoid membranes in intact chloroplasts was investigated by time-resolved optical spectroscopy. The thylakoid transmembrane pH gradient ([delta]pH) was monitored indirectly by measuring the rate of cytochrome (Cyt) f reduction following a light flash of sufficient duration to create a sizable [delta]pH. The results show that the rate of Cyt f reduction is controlled in part by the internal pH of the thylakoid inner aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was maximal, whereas at lower pH values from 6.5 to 5.5 the reduction rate decreased to 25% of the maximal rate. The ability of three uncouplers, nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to accelerate the rate of Cyt f reduction was determined for intact chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the uncouplers for collapsing the [delta]pH was determined using the empirical relationship between the [delta]pH and the Cyt f reduction rate. For intact chloroplasts, nigericin was the most effective uncoupler, followed by carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with bovine serum albumin. Gramicidin D, even at high gramicidin:chlorophyll ratios, did not completely collapse the pH gradient, probably because it partitions in the envelope membranes and does not enter the intact chloroplast.     

Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K_m values for GSSG and NADPH were 23 μm and 4.9 μm, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.     

Photoinhibition of Chloroplast Reactions. II. Multiple Effects

Ultraviolet light inhibits the photoreduction of 2,6-dichlorophenolindo-phenol or nicotinamide adenine dinucleotide phosphate with water as the electron donor (evolution of oxygen) but not the photoreduction of nicotinamide adenine dinucleotide phosphate with ascorbate as the electron donor. It inhibits photophosphorylation associated with either system. Experiments undertaken to test whether plastoquinone is the site of UV inhibition yielded inconclusive results.

Visible light (> 420 mμ) causes the loss of all chloroplast activities, photosystem I being more sensitive than system II. The data suggests 2 modes of action for visible light. The one sensitized by system II results in damage resembling that of UV light. The other, sensitized by system I, results in the destruction of the reaction center of this system.

     

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris. I. Localization of Activity in the Chloroplasts of a Chloroplast Containing Fraction from Developing Leaves

The incorporation of uniformly labeled leucine-¹⁴C into protein by a chloroplast containing fraction from developing primary leaves of bean is reported. Chloroplasts, obtained from week old plants grown in darkness, and then illuminated with white light for 12 hours, were shown to be the principal sites of incorporating activity. Incorporation may continue for 2 hours. Rates of up to 50 μμmole leucine incorporated per mg protein per hour are observed when a 1 hour assay period is used. Incorporation is only partially sensitive to ribonuclease.     

Sulfane-Activated Reduction of Cytochrome c by Glutathione

The inorganic sulfane tetrathionate (^-O₃SSSSO₃^-) resembles glutathione trisulfide (GSSSG) in that it remarkably activates the reduction of cytochrome c by GSH, both under aerobic and anaerobic conditions. These observations can be explained by the formation of the persulfide GSS^-, due to nucleophilic displacements of sulfane sulfur. The GSS^- species has previously been proposed to act as a chain carrier in the catalytic reduction of cytochrome c, and perthiyl radicals GSS·, formed in the reduction step, were thought to recycle to sulfane via dimerization to GSSSSG.² The present study provides some arguments in favour of a chain mechanism involving the GSS· + GS^- ⇄ (GSSSG)^- equilibrium and sulfane regeneration by a second electron transfer from (GSSSG)· ^- to cytochrome c.

Thiosulfate sulfurtransferase (rhodanese) is shown to act as a cytochrome c reductase in the presence of thiosulfate and GSH, and again the generation of GSS^- can be envisaged to explain this result.     

Reversal of Copper Inhibition in Chloroplast Reactions by Manganese

In the Mehler reaction, a Hill reaction utilizing molecular oxygen as the electron acceptor, rates of net oxygen uptake are stimulated by added manganous ions. Both whole cell photosynthesis and the Mehler reaction are inhibited by copper. Copper inhibition of the Mehler reaction can be reversed by manganese salts. Glutathione. which alone has no effect on Mehler reaction rates, enhances the effect of manganese in reversing copper inhibition. The effects of added Cu²⁺, Cu²⁺ and Mn²⁺, or Cu²⁺, Mn²⁺, and glutathione exhibit no induction phenomena when measured manometrically. Furthermore, the order of addition of these factors is unimportant: final rates are dependent only on the composition of reaction mixtures. Compared to the Mehler reaction, conventional Hill reactions are less sensitive to copper poisoning, while certain chloroplast mediated photoxidations (e.g. the photoxidation of diketogulonic acid) are far more sensitive. In all of the chloroplast mediated photoreactions tested, manganese is effective in reducing the sensitivity to copper poisoning.     

Pyrroloquinoline Quinone-Dependent Cytochrome Reduction in Polyvinyl Alcohol-Degrading Pseudomonas sp. Strain VM15C

A polyvinyl alcohol (PVA) oxidase-deficient mutant of Pseudomonas sp. strain VM15C, strain ND1, was shown to possess PVA dehydrogenase, in which pyrroloquinoline quinone (PQQ) functions as a coenzyme. The mutant grew on PVA and required PQQ for utilization of PVA as an essential growth factor. Incubation of the membrane fraction of the mutant with PVA caused cytochrome reduction of the fraction. Furthermore, it was found that in spite of the presence of PVA oxidase, the membrane fraction of strain VM15C grown on glucose without PQQ required PQQ for cytochrome reduction during incubation with PVA. The results provide evidence that PVA dehydrogenase couples with the electron transport chain of PVA-degrading bacteria but that PVA oxidase does not.     

Synthesis of a Putative c-Type Cytochrome by Intact, Isolated Pea Chloroplast

In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea (Pisum sativum L.) chloroplasts were incubated with [¹⁴C]-5-aminolevulinic acid. The major labeled band (M_r = 43 kilodaltons by lithium dodecyl sulfate-polyacrylamide gel electrophoresis) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H₂O₂ stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [¹⁴C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome; however, the possibility that it might be a protein containing a covalently linked linear tetrapyrrole was not ruled out.     

Dichlorophenylurea-insensitive Reduction of Silicomolybdic Acid by Chloroplast Photosystem II

The photoreduction of silicomolybdate and other heteropoly ions by chloroplasts is insensitive to 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU). Both water and diphenylcarbazide can be used as electron source for the reduction. Three different assays for silicomolybdate reduction are described including oxygen evolution, formation of a reduced heteropoly blue silicomolybdate, or an indirect assay for reduced silicomolybdate by redox indicators, such as ferricyanide or cytochrome c. The effects of detergents and tris washing are consistent with silicomolybdate reduction through photosystem II before the DCMU site. The effects of orthophenanthroline and bathophenanthroline indicate chelator-sensitive sites in photosystem II before the site of DCMU action.     

Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58–67 and 100–109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth.     

Oxidation versus Reductive Detoxification of SO(2) by Chloroplasts

Intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) both oxidized and reduced added sulfite in the light. Oxidation was fast only when endogenous superoxide dismutase was inhibited by cyanide. It was largely suppressed by scavengers of oxygen radicals. After addition of O-acetylserine, chloroplasts reduced sulfite to cysteine and exhibited sulfite-dependent oxygen evolution. Cysteine synthesis from sulfite was faster than from sulfate. The results are discussed in relation to species-specific differences in the phytotoxicity of SO₂.