Analysis of the proposed Fe-SX binding region of Photosystem 1 by site directed mutation of PsaA in Chlamydomonas reinhardtii.

The psaA and psaB genes of the chloroplast genome in oxygenic photosynthetic organisms code for the major peptides of the Photosystem 1 reaction center. A heterodimer of the two polypeptides PsaA and PsaB is thought to bind the reaction center chlorophyll, P700, and the early electron acceptors A0, A1 and Fe-SX. Fe-SX is a 4Fe4S center requiring 4 cysteine residues as ligands from the protein. As PsaA and PsaB have only three and two conserved cysteine residues respectively, it has been proposed by several groups that Fe-SX is an unusual inter-peptide center liganded by two cysteines from each peptide. This hypothesis has been tested by site directed mutagenesis of PsaA residue C575 and the adjacent D576. The C575D mutant does not assemble Photosystem 1. The C575H mutant contains a photoxidisable chlorophyll with EPR properties of P700, but no other Photosystem 1 function has been detected. The D576L mutant assembles a modified Photosystem 1 in which the EPR properties of the Fe-SA/B centers are altered. The results confirm the importance of the conserved cysteine motif region in Photosystem 1 structure.

Mode of Action Studies on a Chiral Diphenyl Ether Peroxidizing Herbicide: Correlation between Differential Inhibition of Protoporphyrinogen IX Oxidase Activity and Induction of Tetrapyrrole Accumulation by the Enantiomers.

The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-O-(acetic acid, methyl ester) (DPEI) induced an abnormal accumulation of protoporphyrin IX in darkness in the green alga Chlamydomonas reinhardtii, as determined by high-performance liquid chromatography and spectrofluorimetry. It also inhibited the increase in cell density of the alga in light-grown cultures with an I(50) (concentration required to decrease cell density increase to 50% of the noninhibited control value) of 0.16 mum. The relative ability of four peroxidizing diphenyl ether herbicides to cause tetrapyrrole accumulation in C. reinhardtii correlated qualitatively with their ability to inhibit the increase in cell density in light-grown cultures. The purified S(-) enantiomer of the optically active phthalide DPE 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methylphthalide (DPEIII), which has greater herbicidal activity than the R(+) isomer, induces a 4- to 5-fold greater tetrapyrrole accumulation than the R(+) isomer. The I(50) for inhibition of increase in cell density in light-grown cultures of C. reinhardtii by the S(-) isomer (0.019 mum) is less than 25% of that for the R(+) isomer. DPEIII inhibits protoporphyrinogen IX oxidase activity in pea (Pisum sativum) etioplast lysates, with the S(-) enantiomer showing considerably greater potency than the R(+) isomer and the racemic mixture showing a potency intermediate between the two. The results indicate that the site at which DPEs inhibit protoporphyrinogen IX oxidase shows chiral discrimination and provide further evidence for the link between inhibition of this enzyme, protoporphyrin IX accumulation, and the phytotoxicity of DPE herbicides.

An e.x.a.f.s. study of the manganese O2-evolving complex in purified Photosystem II membrane fractions. The S1 and S2 states.

Manganese K-edge X-ray spectra have been obtained for Photosystem II samples isolated using Triton X-100 detergent and samples further purified by n-heptyl beta-D-thioglucoside detergent treatment to remove light-harvesting polypeptides and low-affinity calcium. The structure of the manganese complex is very similar for the two detergent preparations used. Analysis of the e.x.a.f.s. spectra for samples in the S1 and S2 states indicate changes in bond lengths for the shells of oxygen/nitrogen atoms. For the S1 state, oxygen shells at 0.181 and 0.193 nm (1.81 and 1.93 A) were observed and one manganese shell at 0.270 nm (2.70A). In the S2 state the oxygen bond lengths are longer at 0.184 and 0.200 nm (1.84 and 2.00 A). Additionally a shell of scatterers at 0.37 nm (3.7 A) was observed in both states which could be fitted to models with calcium scatterers at this distance.

Investigation of the origin of the "S3" EPR signal from the oxygen-evolving complex of photosystem 2: the role of tyrosine Z.

The origin of the "S3" EPR signal from calcium-depleted photosystem 2 samples has been investigated. This signal is observed after freezing samples under illumination and has been assigned to an interaction between the manganese cluster and an oxidized histidine radical [Boussac et al. (1990) Nature 347; 303-306]. In calcium-depleted samples prepared by three different methods, we observed the trapping of the tyrosine radical YZ+ under conditions which also formed the "S3" signal. An "S3"-type signal and YZ+ were also formed in PS2 samples treated with the water analogue ammonia. Following illumination at 277 K, the "S3" and YZ+ signals decayed at the same rate at 273 K in the dark. Both the YZ+ and "S3" signals decayed on storage at 77 K and could be subsequently regenerated by illumination at 8-77 K. No evidence to support histidine oxidation was found. The effects of DCMU, chelators, and alkaline pH on the dark-stable multiline S2 and the "S3" signals from calcium-depleted samples were determined. Both signals required the presence of EGTA or citrate for maximum yield. The addition of DCMU caused a reduction in the yield of "S3" generated by freezing under illumination. Incubation at pH 7.5 resulted in the loss of both signals. We propose that a variety of treatments which affect calcium and chloride binding cause a stabilization of the S2 state and slow the reduction of YZ+. This allows the trapping of YZ+, the interaction with the manganese cluster (probably in the S2 state) resulting in the "S3" signal. The data allow the position of the manganese cluster to be estimated as within 10 A of tyrosine Z (D1-161).

Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids.

The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity.

Increased age affects properties characterizing behavioral plasticity in freely behaving Aplysia.

In the marine mollusc Aplysia, in vitro studies showed that the gill withdrawal reflex (GWR) and its neuronal substrates were altered by age. In contrast, age minimally affected the gill respiratory pumping movements (GPM) and its neuronal substrates. Based on the respective properties of the GWR- and GPM-pathways in vitro, we proposed that the more pronounced the effect of age, the greater the expression of plasticity in a pathway. This conclusion may hold for in vitro preparations, but it remained to be demonstrated in intact animals. Based on this conclusion, the GWR should exhibit greater plasticity than the GPM in intact animals. Using freely behaving Aplysia, we tested for plasticity of the GWR and the GPM in three age groups (young, mature, and old). The tests for behavioral plasticity were: Graded responses to varying stimulus strength, response decrement (or habituation) to repetitive stimulation, enhanced response to dishabituating stimuli, and the effect of the GWR stimulus on the GPM and the GPM stimulus on the GWR. The GWR in mature animals exhibited all four properties, but in old animals, graded responses and habituation were significantly altered and in young animals habituation and dishabituation were absent. The GPM exhibited fewer of the properties than the GWR, only graded responses and response decrement, both of which were generally the same in the three groups. We found that behavioral plasticity and age-induced plasticity are related in freely behaving animals and are consistent with in vitro findings. The effect of age on properties characterizing plasticity at both the behavioral and pathway levels is discussed.

Mode of Action Studies on Nitrodiphenyl Ether Herbicides : II. The Role of Photosynthetic Electron Transport in Scenedesmus obliquus.

The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-o-(acetic acid, methyl ester) (DPEI) induces light- and O(2)-dependent lipid peroxidation and chlorophyll (Chl) bleaching in the green alga Scenedesmus obliquus. Under conditions of O(2)-limitation, these effects are diminished by prometyne and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), both inhibitors of photosynthetic electron transport. Mutants in which photosynthetic electron transport is blocked are also resistant to DPEI under conditions of O(2)-limitation. Light- and O(2)-dependent lipid peroxidation and Chl bleaching are also induced by 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methoxyphthalide (DPEII), a diphenyl ether whose redox properties preclude reduction by photosystem I. However, these effects of DPEII are also inhibited by DCMU. Under conditions of high aeration, DCMU does not protect Scenedesmus cells from Chl bleaching induced by DPEI, but does protect against paraquat. DPEI, but not paraquat, induces tetrapyrrole formation in treated cells in the dark. This is also observed in a mutant lacking photosystem I but is suppressed under conditions likely to lead to O(2) limitation. Our results indicate that, in contrast to paraquat, the role of photosynthetic electron transport in diphenyl ether toxicity in Scenedesmus is not to reduce the herbicide to a radical species which initiates lipid peroxidation. Its role is probably to maintain a sufficiently high O(2) concentration, through water-splitting, in the algal suspension.