Evidence that a Chloroplast Surface Protein Is Associated with a Specific Binding Site for the Precursor to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase.

Most chloroplast proteins are encoded by nuclear genes and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported posttranslationally into the chloroplasts, where they are proteolytically processed, and sorted to their proper locations. The first step of this import process is thought to be the binding of precursors to putative receptors on the outer envelope membrane of chloroplasts. We have investigated the interaction of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with its putative receptor by using a heterobifunctional, photoactivatable cross-linker. The resulting cross-linked conjugate has a molecular weight of 86,000, and is present on the surface of chloroplasts as determined by its sensitivity to digestion with protease. Control experiments demonstrated that the label in the conjugate is derived from small subunit precursor and that the conjugate is formed only when modified precursor is reacted in the presence of chloroplasts. Based on these results, we postulate that a protein on the surface of chloroplasts is part of the receptor which interacts with the small subunit precursor.

Mass spectrometrically derived amino acid sequence of thioredoxin from Chlorobium, an evolutionarily prominent photosynthetic bacterium.

The amino acid sequence of the thioredoxin isolated from the photosynthetic green sulfur bacterium Chlorobium thiosulfatophilum was determined chiefly by fast atom bombardment mass spectrometry combined with Edman degradation and tandem mass spectrometry. For this purpose, the protein was digested with trypsin, alpha-chymotrypsin, thermolysin, and Staphylococcus aureus protease or combinations thereof. Chemical cleavage with cyanogen bromide was also used alone or in combination with trypsin. The resulting sequence of 108 amino acids is as follows: Ala-Gly- Lys-Tyr-Phe-Glu-Ala-Thr-Asp-Lys-Asn-Phe-Gln- Thr-Glu-Xle-Xle-Asp-Ser-Asp-Lys-(Ala-Val)-Xle- Val-Asp-Phe-Trp-Ala-Ser-Trp-Cys-Gly-(Pro-Cys)- Met-Met-Xle-Gly-Pro-Val-Xle-Glu-Gln-Xle-Ala-Asp- Asp-Tyr-Glu-Gly-Lys-Ala-Xle-Xle-Ala-Lys-Xle-Asn- Val-Asp-Glu-Asn-Pro-Asn-Xle-Ala-Gly-Gln-Tyr-Gly- Xle-Arg-Ser-Xle-Pro-Thr-Met-Xle-Xle-Xle-Ly s- (Gly-Gly-Lys)-Val-Val-Asp-Gln-Met-Val-Gly-Ala- Xle-Pro-Lys-Asn-Met-Xle-Ala-Lys-Lys-Xle-Asp-Glu-His-Il e-Gly (where Xle represents leucine or isoleucine; sequences in parentheses are based on homology considerations). It exhibits less than 53% homology with Escherichia coli thioredoxin.

Evidence for a nickel-containing carbon monoxide dehydrogenase in Methanobrevibacter arboriphilicus.

In growing cultures of Methanobrevibacter arboriphilicus (Methanobrevibacter arboriphilus), the synthesis of active carbon monoxide dehydrogenase required nickel. The 21-fold-purified enzyme from 63Ni-labeled cells of M. arboriphilicus comigrated with 63Ni during gel filtration. These results provide evidence that the carbon monoxide dehydrogenase of methanogens is a nickel protein.

Ferredoxin/flavoprotein-linked pathway for the reduction of thioredoxin.

Thioredoxins are small redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of important biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Reduced ferredoxin is known to serve as the source of reducing power for the reduction of thioredoxins only in photosynthetic cells that evolve oxygen. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction is considered to be NADPH. We now report evidence that Clostridium pasteurianum, an anaerobic bacterium normally living in the soil unexposed to light, resembles photosynthetic cells in that it uses reduced ferredoxin as the reductant for thioredoxin. Moreover, the transfer of electrons from reduced ferredoxin to thioredoxin is catalyzed by a flavoprotein enzyme that has not been detected in other organisms. Our results reveal the existence of a pathway for the reduction of thioredoxin in which ferredoxin, reduced fermentatively either by molecular hydrogen or by a carbon substrate, provides the reducing power for the flavoprotein enzyme ferredoxin-thioredoxin reductase, which in turn reduces thioredoxin.

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach.

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of (14)CO(2) under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO(2) concentrations. Incorporation of (14)C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [(14)C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO(2) uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased (14)C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased (14)CO(2) labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net (14)CO(2) fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase (14)CO(2) incorporation by 15% in isolated mesophyll cells under air levels of CO(2).

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells.

The effects of added glycine hydroxamate on the photosynthetic incorporation of (14)CO(2) into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO(2) and 20% O(2)) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO(2) and 2.7% O(2)). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO(2) and NH(4) (+). During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the "reverse" flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.

Effects of carbon dioxide and oxygen on the regulation of photosynthetic carbon metabolism by ammonia in spinach mesophyll cells.

Photosynthetic carbon metabolism of isolated spinach mesophyll cells was characterized under conditions favoring photorespiratory (PR; 0.04% CO(2) and 20% O(2)) and nonphotorespiratory (NPR; 0.2% CO(2) and 2% O(2)) metabolism, as well as intermediate conditions. Comparisons were made between the metabolic effects of extracellularly supplied NH(4) (+) and intracellular NH(4) (+), produced primarily via PR metabolism. The metabolic effects of (14)CO(2) fixation under PR conditions were similar to perturbations of photosynthetic metabolism brought about by externally supplied NH(4) (+); both increased labeling and intracellular concentrations of glutamine at the expense of glutamate and increased anaplerotic synthesis through alpha-ketoglutarate. The metabolic effects of added NH(4) (+) during NPR fixation were greater than those during PR fixation, presumably due to lower initial NH(4) (+) levels during NPR fixation. During PR fixation, addition of ammonia caused decreased pools and labeling of glutamate and serine and increased glycolate, glyoxylate, and glycine labeling. The glycolate pathway was thus affected by increased rates of carbon flow and decreased glutamate availability for glyoxylate transamination, resulting in increased usage of serine for transamination. Sucrose labeling decreased with NH(4) (+) addition only during PR fixation, suggesting that higher photosynthetic rates under NPR conditions can accommodate the increased drain of carbon toward amino acid synthesis while maintaining sucrose synthesis.

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2).

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO(2) fixation for more than 60 hours. The incorporation of (14)CO(2) under saturating CO(2) conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for gamma-aminobutyric acid. The measurements of specific radio-activities and of the approaches to (14)C "saturation" of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.Added ammonia decreased carbon fixation into carbohydrates and increased fixation into carboxylic acids and amino acids. Different amino acids were, however, affected in different and highly specific ways. Ammonia caused large stimulatory effects in incorporation of (14)C into glutamine (a factor of 21), aspartate, asparagine, valine, alanine, arginine, and histidine. No effect or slight decreases were seen in glycine, serine, phenylalanine, and tyrosine labeling. In the case of glutamate, (14)C labeling decreased, but specific radioactivity increased. The production of labeled gamma-aminobutyric acid was virtually stopped by ammonia.The results indicate that added ammonia stimulates the reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase, as seen with other plant systems. The data on the effects of added ammonia on total labeling, pool sizes, and specific radioactivities of several amino acids provides a number of indications about the intracellular sites of principal synthesis from carbon skeletons of these amino acids and the selective nature of effects of increased intracellular ammonia concentration on such synthesis.

Effects of ammonia on carbon metabolism in photosynthesizing isolated mesophyll cells from Papaver somniferum L.

Addition of ammonia to a suspension of photosynthesizing isolated mesophyll cells from P. somniferum quantitatively alters the pattern of carbon metabolism by increasing rates of certain key ratelimiting steps leading to amino-acid synthesis and by decreasing rates of rate-limiting steps in alternative biosynthetic pathways. Of particular importance is the stimulation of reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase. The increased rates of these two reactions, which result in an increased flow of carbon into the tricarboxylic-acid cycle, correlate with a rapid rise in glutamine (via glutamine synthetase) which draws carbon off the tricarboxylic-acid cycle as α-ketoglutarate. Increased flux of carbon in this direction appears to come mainly at the expense of sucrose synthesis. The net effect of addition of ammonia to mesophyll cells is thus a redistribution of newly fixed carbon away from carbohydrates and into amino acids.