Hydrogenase from Chlamydomonas reinhardtii was purified to homogeneity by five columnchromatography deps under strict anaerobic conditions. The cells were disrupted by mild treatment with detergent. The enzyme was purified 6100-fold, resulting in a specific activity for H, evolution of 935 pmol . min-' . mg protein-' at 25"C, using reduced methyl viologen as electron donor. The optimal temperature for hydrogen evolution is 60°C, the optimal pH value is 6.9. The K, value for methyl viologen is 0.83 mM, for ferredoxin, 35 pM. From SDSFAGE gels, the protein was judged to be pure. On non-denaturing gels, run under nitrogen, a single band was detected after activity staining. This band corresponded to the single band observed on denaturing SDS gels, which had an apparent molecular mass of 48 m a . If the band was cut out of the native gel and incubated with reduced methyl viologen, hydrogen evolution could be measured. The purified enzyme contains 4Fe atorns/mol. The amino acid composition and the N-terminal amino acid sequence (24 residues) of the protein were determined. No significant amino acid sequence homologies could be found to any sequences from prokaryotic hydrogenases.Many prokaryotes and several eukaryotes have in common an enzyme complex catalyzing the reversible reaction 2 H + + 2 e = H 2 .

The hydrogenase enzyme occurring in Chlamydomonas reinhardtii is induced by anaerobic adaptation of the cells. In aerobically growing cells, antibodies against the hydrogenase failed to detect either active or inactive enzyme. However, already 10 min after the onset of anaerobic adaptation, the protein could be detected. The maximal amount of enzyme was reached after 2-3 hours anaerobiosis. Addition of nickel or iron to the growth medium did not influence activity. In atomic absorption experiments, a Ni/Fe ratio of about 1 : 250 was measured. We, therefore, propose the hydrogenase from C. reinhardtii to be of the Fe-only type. Adaptation in the presence of uncouplers of phosphorylation showed this process to be energy-dependent. From protein synthesis inhibition experiments, it is concluded that the protein is synthesized on cytoplasmic ribosomes and, therefore, must be nuclear encoded. After isolation of intact chloroplasts from adapted cells, the active enzyme was shown, by Western-blotting analysis, to be located in the chloroplasts.Hydrogenases are enzyme complexes, consisting of one or more subunits. They catalyze the reversible reduction of protons to molecular hydrogen as follows:Hydrogenases are found in many prokaryotes, as well as in several eukaryotes. The enzymes were identified in and isolated from a large number of bacteria [l -61, including photosynthetic purple bacteria [7 -91, and cyanobacteria [lo, 111. However, only a few eukaryotic organisms possess hydrogenase activity. In a previous paper, we described purification to homogeneity of hydrogenase from Chlamydomonas reinhardtii [12]. A structurally different enzyme, also showing hydrogen evolution after anaerobic adaptation, was purified from Scenedesmus obliquus [13] Trebst, on the occasion of his 65th birthday.genase uptake activity was found in aerobically growing cells under manganese-deficient conditions. Kessler suggested that quinones, reduced by electrons resulting from hydrogen oxidation, might help prevent damage by photooxidation of chlorophyll [19]. This mechanism may be supported by the recent finding that the membrane-bound hydrogenase from Brudyrhizobium japonicum efficiently catalyzes H, : ubiquinone-1 oxidoreductase activity [20]. Whether this pathway could also function in C. reinhardtii remains an open question, since, in contrast to the bacterial enzyme, the algal hydrogenase does not contain nickel and, therefore, probably possesses a structurally different reactive center, and the activity of the algal enzyme in H, evolution is much higher, whereas the enzyme from Brudyrhizobium is a typical H,-uptake enzyme. The enzyme plays a key role in hydrogen metabolism in bacteria, but in algae its function is limited. The first observation of hydrogen metabolism in algae was made by Gaffron [21, 221. Upon illumination of anaerobically adapted cells, he observed CO, reduction accompanied by uptake of molecular H,. This process was named photoreduction. Hydrogen production was first measured in a green alga, Scenedesmus obliquus, by...

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It is concluded that the "on" kinetics o f a com pound to its binding environment at the D 1 protein are determined principally by the accessibility o f the niche to the com pound. The dif ferences in activity between herbicides are mainly due to variations in the release kinetics.

In Photosystem II (PS II), water is oxidized to molecular oxygen and plastoquinone is reduced to plastoquinol. The oxidation of water requires the accumulation of four oxidizing equivalents, through the so-called S-states of the oxygen evolving complex; the production of plastoquinol requires the accumulation of two reducing equivalents on a bound plastoquinone, QB. It has been generally believed that during the flash-induced transition of each of the S-states (Sn → Sn+1, where n=0, 1, 2 and 3), a certain small but equal fraction of the PS II reaction centers are unable to function and, thus, 'miss' being turned over. We used thoroughly dark-adapted thylakoids from peas (Pisum sativum) and Chenopodium album (susceptible and resistant to atrazine) starting with 100% of the oxygen evolving complex in the S1 state. Thylakoids were illuminated with saturating flashes, providing a double hit parameter of about 0.07. Our experimental data on flashnumber dependent oscillations in the amount of oxygen per flash fit very well with a binary pattern of misses: 0, 0.2, 0, 0.4 during S0 → S1, S1 → S2, S2 → S3 and S3 → S0 transitions. Addition of 2 mM ferricyanide appears to shift this pattern by one flash. These results are consistent with the 'bicycle' model recently proposed by V. P. Shinkarev and C. A. Wraight (Oxygen evolution in photosynthesis: From unicycle to bicycle, 1993, Proc Natl Acad Sci USA 90: 1834-1838), where misses are due to the presence of P(+) or QA (-) among the various equilibrium states of PS II centers.