The Sfreptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450sul (CYP105Al) was expressed in transgenic tobacco (Nicofiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450sul antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450sul-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl~-1,2-benzoisothiazole-7-sulfonamide-1,l-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450sul are susceptible to injury from R7402 treatment that is harmless to plants without P45OSul. Thus, P450sul expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450sul from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450sul from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent.P450 monooxygenases play a central role in plant response to a variety of environmental challenges, including herbicide and other pesticide treatments (Cole, 1983;Durst, 1991). The best examples of this role are those P450s that can chemically alter an herbicide to a form with reduced phytotoxicity, resulting in much higher herbicide tolerance in the plant where this metabolism occurs (Frear et al., 1969(Frear et al., ,1991Sweetser et al., 1982; Jacobson and Shimabukuru, 1984;Brown, 1990). Significant variability in P450 enzyme activity exists among plant species, both in the presence of distinct enzymic forms and in their range of substrate specificity. This variability is a major determinant in herbicide selectivity, the differential herbicide sensitivity of weed and crop species. Altering herbicide selectivity, particularly confemng resistance in a crop plant, might be accomplished by expression of Present address:

Hydrogenase, purified to an average specific activity of 328 jsmol of H2 evolved/(min X mg of protein) from Clostridium pasteurianum W5, was found to have 4-5 Fe and 4-5 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 Fe per molecule. Displacement of the iron-sulfur cluster from hydrogenase by thiophenol in 80% hexamethyl phosphoramide:20% H20 yielded the Fe4S4 (thiophenyl)4 dianion according to absorption spectroscopy. Electron paramagnetic resonance spectroscopy at 12 K showed that the iron-sulfur cluster in the enzyme could be reduced by the H2 to a state (g-values of 2.098, 1.970, and 1.898) similar to that in reduced ferredoxin and could be oxidized by dichlorophenolindophenol or H+ to a state (gvalues at 2.099, 2.041, and 2.001) similar to that in high potential iron-sulfur proteins. These oxidations and reductions appeared to occur within the turnover time of the enzyme. Deuterium failed to narrow the electron paramagnetic resonance signal in either state, but the competitive inhibitor carbon monoxide reversibly formed a compound with either state and substantially altered the electron paramagnetic resonance. 13CO produced a broadening of these signals, suggesting the formation of a direct CO complex with the iron-sulfur cluster. These data are consistent with a model of the active site of the enzyme in which a four-iron four-sulfur cluster is a component that can accept one or two electrons from and donate either one or two electrons to substrates, and in which the iron-sulfur cluster serves as the site of binding of gaseous ligands. Hydrogenases have been purified extensively from C. pasteurianum (1-3), Desulfovibrio vulgaris (4, 5), and Chromatium (6). Although it has been known for some time that the enzyme is an iron-sulfur enzyme (ref. 7, and references therein), no evidence has appeared so far about the nature of the iron-sulfur center nor about its mode of action in the enzyme. In this communication we report evidence for the nature of the iron-sulfur cluster, its oxidation states during turnover, and its interaction with the competitive inhibitor carbon monoxide. A hypothesis emerges which suggests further useful avenues of study. MATERIALS -AND METHODSMethyl viologen from Koch-Light Laboratories (Colnbrook, England) was recrystallized twice from ethanol-acetone and analyzed (found 56.04% C, 5.50%

02 irreversibly inactivates hydrogenase from Chlamydomonas reinhardiThe mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of 02 (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres-' minute-1 (1.4 x 105 molar-' minute-1). At levels above 0.01 atmosphere 02, the increased numbers of 02 molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one 02 molecule and resulting in lowered rates of inactivation.CO is a reversible inhibitor of hydrogenase acting competitively against H2. The Ki for CO is 0.0010 atmosphere. CO antagonizes 02 inactivation. In a period when complete inactivation by 02 would usualHy occur, the presence of CO greatly reduces the inactivation rate.After 3 hours of adaptation in whole cells, the presence of H2 lowers the rate of deadaptation of hydrogenase. Inasmuch as H2 promotes increased 02 uptake the cellular concentration of 02 is likely to be lower. After 48 hours of adaptation 02 uptake is reduced even when H2 is present and the pattern of deadaptation under 02 with and without H2 and CO is qualitatively the same as observed for the inactivation of cell-free hydrogenase. The mechanism of inactivation of celi-free hydrogenase by 02 may be the same as the mechanism for loss of hydrogenase during deadaptation in whole algal cells.Since the initial observation of H2 metabolism in green algae the sensitivity of algal H2 metabolism to O2 has been noted (5). Simultaneous uptake of H2 and O2 (the oxyhydrogen reaction) is inhibited when the O2 level exceeds a specific concentration (6, 10). Also, the photoproduction of 02 in algae will cause cessation of H2 metabolism. Removal of photoproduced O2 reversed the inhibition of hydrogenase (9) C. moewusii was grown in minimal medium plus acetate (7).The hydrogenase was prepared as above.Assays. Protein was assayed by the biuret method (8) and Chl was assayed spectrophotometrically in 80% acetone (2). The enzyme was assayed by MV3 reduction followed on a Gilford recording spectrophotometer at 605 nm. Hydrogenase solution was injected into a cuvette with 2.0 ml of anaerobically prepared 10 mM MV in 50 mm Tris-HCl (pH 8.0) under 1.0 atm H2 at 25 C. The extinction coefficient for reduced MV was determined to be 9.6 mM-' cm-' by reduction with excess sodium dithionite at pH 8.0. One unit of hydrogenase activity is defined as the reduction of 1.0 mol MV/min.

Using manometric and enzymic techniques, H2 and CO2 evolution in darkness and light has been studied in the green alga Chianydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain.In the dark, starch was broken down, and H2 and CO2 was released. The uncoupler, carbonyl cyanide m-fluorophenylhydrazone with an optimum concentration of 5 to 10 micromolar, increased the rate of CO2 release and starch breakdown but depressed H2 formation. It m-fluorophenylhydrazone, stimulated H2 photoproduction by removing ATP which limited the sequence of reactions. The contribution of photosystem II to the photoproduction of H2, as judged from the effect of 10 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, was at least 80%.CO2 photoevolution increased Unearly with time, but H2 photoevolution occurred in two phases: a rapid initial phase folowed by a second slower phase. The rate of H2 release increased hyperbolically with light intensity, but the rate of CO2 production tended to level off and decrease with increasing light intensity, up to 145 watts per square meter. It was proposed that a changing CO2 and H2 ratio is the result of interaction between the carbon and hydrogen metabolism and the photosynthetic electron transport chain.Studies with chlorophyllous algae have established that H2 evolution in the light is the result of electron transport through the photosystems associated with an adaptable hydrogenase. These algae also produce H2 in the dark but at a lower rate. Two mechanisms have been proposed to account for H2 release in the light, while the pathway for the dark release of H2 has received 'Supported by Department of Energy (10-EY-76-5-02-3231) and National Science Foundation (PCM 79-22612 light (3, 14). It is known that gas evolution is stimulated by added glucose (5). Data obtained with position-labeled glucose led Kaltwasser et al. (13) to propose that classical glycolysis in Scenedesmus obliquus is the pathway involved. From a stoichiometric analysis of the products-which included ethanol, glycerol, and acetate, in addition to CO2 and H2-Klein and Betz (15) also proposed glycolysis for the heterofermentative breakdown of the reserve substance, starch, in Chlamydomonas reinhardii. This suggestion was supported by a similar fermentation pattern in Chlorella vulgaris (22). On the basis of a depression of H2 evolution in the dark by uncouplers of phosphorylation, Gaffron and Rubin (5) suggested that the fermentation yielded not only CO2 but also ATP. ATP would be required to raise the redox potential of the electrons from the reductant (NADH) to a higher one necessary for H2 production. In contrast to inhibiting H2 evolution in the dark, the uncoupler elevated the photorelease of H2 and CO2. Acetate has been reported to stimulate H2 evolution insensitive to DCMU, and Healy (10) suggested an anaerobically functioning citric acid cycle in Chlamydomonas moewusii to explain this observation. In his formu...

No abstract