No abstract

A study, using ab initio quantum chemical methods, of the first step in the reaction mechanism of Rubisco, the enolization of the substrate, ribulose bisphosphate, is reported. This is the first such study that takes into account the likely roles of critical features within the active site. On the basis of molecular dynamics relaxation of the complex between activated enzyme and substrate using X-ray crystallographic structures as starting coordinates, a 29-atom fragment that mimicked the active site was constructed. States along a proposed reaction pathway were calculated using density functional theory and Moller-Plesset second-order perturbation theory. The results are consistent with the postulate that the base that abstracts the C3 proton of ribulose bisphosphate is the metal-stabilized carbamate of Lys-201 formed during the activation process. The calculations suggest that the active-site residue, Lys-175, is charged before enolization commences and they indicate a possible means by which the enzyme directs the incoming CO2 to attack the C2 carbon atom of the enediol, rather than the chemically very similar C3 atom.

Fully functional Synechococcus PCC 6301 ribulose 1,5-bisphosphate carboxylase-oxygenase (kcat = 11.8 s-1) was assembled in vitro following separate expression of the large- and small-subunit genes in different Escherichia coli cultures. The small subunits were expressed predominantly as monomers, in contrast to the large subunits which have been shown to be largely octameric when expressed separately [Andrews, T. J. (1988) J. Biol. Chem. 263, 12213-12219]. This separate expression system was applied to the study of mutations in the amino-terminal arm of the small subunit, which is one of the major sites of contact with the large subunit in the assembled hexadecamer. It enabled the effects of a mutation on the tightness of binding of the small subunit to the large-subunit octamer to be distinguished from the effects of the same mutation on catalysis carried out by the assembled complex when fully saturated with mutant small subunits. This important distinction cannot be made when both subunits are expressed together in the same cell. Substitutions of conserved amino acid residues at positions 14 (Ala, Val, Gly, or Asp instead of Thr) and 17 (Cys instead of Tyr), which make important contacts with conserved large-subunit residues, were introduced by site-directed mutagenesis. All mutant small subunits were able to bind to large subunits and form active enzymes. A potential intersubunit hydrogen bond involving the Thr-14 hydroxyl group is shown to be unimportant. However, the binding of Gly-14, Asp-14, and Cys-17 mutant small subunits was weaker, and the resultant mutant enzymes had reduced catalytic rates compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

The 813C values of several seagrasses were considerably less negative than those of terrestrial C3 plants and tended The marine monocotyledonous angiosperms, the seagrasses, have many interesting adaptations to their submerged environment (9,18). One of these is the adaptation of their photosynthetic physiology to enable the direct assimilation of inorganic carbon from seawater. The leaf anatomy of seagrasses is very different from that of terrestrial plants. The leaves have no stomata, and almost all of the chloroplasts are located in a single layer of thick walled epidermal cells. The mesophyll is comprised of highly vacuolated cells, containing very few chloroplasts, interspersed with large gas lacunae (2, 10). Evidence exists that the lacunal gas may become enriched in 02, relative to atmospheric levels, during periods of active photosynthesis (11). It is possible that the photosynthetic apparatus of seagrasses may be exposed to higher than atmospheric 02 concentrations. This situation is known to promote photorespiration (6) and is rarely, if ever, encountered by terrestrial plants in the natural environment.The photosynthetic pathway used by seagrasses to fix carbon is of interest. This interest has been stimulated by reports that the carbon isotope ratios of seagrasses resembled those of C4 plants rather than C3 plants and that C4 acids were labeled early during 14C fixation by Thalassia testudinum (1)(2)(3) 10 MATERIALS AND METHODSPlant Material. Both species were collected near Magnetic Island, North Queensland. T. hemprichii (Ehrenb.) Aschers was collected from an intertidal reefflat in Cockle Bay and H. spinulosa (R. Br.) Aschers from a subtidal location at 5-to 10-m depth off Nobby Head. Leafy shoots with attached rhizomes and roots were transported to the laboratory, stored in aerated seawater in the dark overnight, and used the following day. Only young leaves, free from visible epiphytes, were used for experiments. Segments of the youngest mature T hemprichii leaves (1.5-2.0 cm long) or groups of 8 to 10 leaves at the tips of H. spinulosa branches were selected.Medium for "4C-labeling. Millipore-filtered seawater was buffered with N,N-bis(2-hydroxyethyl)glycine (final concentration, 20 mM), acidified with HCI to below pH 4 and purged overnight with humidified C02-free air at 25 C. The pH was adjusted to 8.22 with carbonate-free NaOH and then NaH14CO3 was added to a final concentration of 2.2 mm with a specific radioactivity of 26 ,uCi ,Imofl' for T. hemprichii experiments and 9 liCi tmol' for H. spinulosa experiments.14C-labeling Procedure. Experiments were conducted at 25 C in a shaking water bath (about 50 oscillations/min). Light (about 100 w m-2) was provided from a mercury vapor lamp, filtered through 10 cm of water. Leaf material was preilluminated for at least 30 min in Millipore-filtered seawater or in nonradioactive medium equivalent in all other respects to the labeling medium. It was then quickly blotted to remove adhering unlabeled medium which would otherwise decrease the s...

Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracca L.) chloroplasts which exhibited very high rates of photosynthetic CO2 reduction and were insensitive to additions of photosynthetic intermediates when CO2 was available at saturating concentrations. Photosynthetic rates were measured polarographically as 02 evolution, and the extent of the reduction of substrate was estimated from the amount of 02 evolved. With CO2 as substrate, inhibition of photosynthesis by 02 was dependent on pH. At pH values above 8, rates of 02 evolution were strongly inhibited by 02 and only a fraction of the added bicarbonate was reduced before 02 evolution ceased. The extent of 02 evolution declined with increasing 02 concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high 02 levels, but the extent of 02 evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic 02 evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and 02 concentration. In contrast, there was little effect of 02 and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by 02. The 23). Oxygenation of RuDP and CO2 evolution from the reaction products would decrease the yield of photosynthesis. Glycolate formation related to the 02 inhibition of photosynthesis has also been explained by oxidative interactions with C6-or Crsugarphosphates (9,16,22).It has been shown that photosynthetic CO2 reduction by isolated chloroplasts is also inhibited by 02 (8,9,21). In the present work, we have studied the effect of 02 on photosynthesis by intact chloroplasts, capable of very high rates of photosynthesis. The aim of our experiments was to gain further insight into the problem of 02 inhibition of photosynthesis in a system less complex than the intact leaf and more sophisticated than a simple enzyme system. MATERIALS AND METHODSSpinach Hybrid 102 (Spinacia oleracea L.) was purchased from Arthur Yates and Co., Sydney, who obtained it from Alf. Christianson Seed Co., Mount Vernon, Wash. Seeds were germinated in vermiculite and either transferred to hydroponic culture or grown in vermiculite. The nutrient solution was the same as used by Tsui (24), except that sequestrene iron chelate (15.4 mg/l) replaced iron tartrate. The plants were grown for 3 to 4 weeks before the leaves (length 9-10 cm) were harvested. The detached leaves were preilluminated for 5 to 10 min at 4 C in white light (11) and chloroplasts were isolated by a modification (11) of the method of Jensen and Bassham (18).Photosynthetic 02 evolution was measured polarographically at 20 C, using CO2, PGA, or OAA as substrate. Chloroplasts (100-200 ,pg Chl) were suspended in 2.5 ml of a buffer containing 300 mm sorbitol, I mM MgCI2, I mm MnCI2, 2 mM EDTA, 10 mM NaCI, 0.8 mm inorganic phosphate (KH2PO4), and 50 mM HEPES, pH 6.8 to 8.5. CO2 was remo...