, 1995). Chlorophyll a (Chla) was determined

from methanol extracts of washed cells using the method of Mackinney (1941). Cultures for 15N2 incorporation analysis were grown and washed as described above and resuspended in AA/8 containing 50 mM fructose, 5 mM glucose, and 50 μM DCMU. For each 15N2 incorporation analysis, duplicate samples of 10 mL of washed cells were added to Hungate tubes, capped, and sparged for 2 min with argon. After 2 min of sparging with argon, 30 U of glucose oxidase and 500 U of catalase were added to each sample via a hypodermic needle (to remove oxygen found to be present in the 15N2 that was subsequently injected) and sparging was continued for 13 min. Then, 4 mL of headspace gas was removed and replaced with 4 mL buy Veliparib of 15N2 (98 atom %15N, Sigma). Samples were incubated at 30 °C with shaking and illumination at 90–100 μE m−2 s−1 for 4 or 7.5 h. H2 production (4 and 7 h) and acetylene reduction (7 h) were measured as described above Z-VAD-FMK cost to determine nitrogenase activity, and the cells were harvested, dried, weighed (generally between 1.5 and 2 mg), and sent to the stable isotope facility at the University of California, Davis, for 15N isotope analysis

by isotope ratio MS. The increase in the percentage of 15N in samples from the 4-h time point to the 7.5-h time point was used as the measure of the rate of 15N2 fixation. At 4–7 h after anaerobic induction in the absence of fixed nitrogen, only the Nif2 nitrogenase functions because no heterocysts are present (Thiel Tolmetin et al., 1997; Weyman et al., 2008). According to the standard equation for the Mo-nitrogenase reaction (N2+8 e−+16 ATP+8 H+2 NH3+16 ADP+16 Pi+H2), a minimum of one-quarter of all electrons used in the reduction reaction results in the production of H2. When the substrate N2 is absent in an argon atmosphere,

all the electrons are used in the reduction of protons to H2 (Barney et al., 2004). We observed that the rates of H2 production in an argon atmosphere were approximately the same in the wild-type (FD) and the three mutant strains: about 100 nmol H2 μg−1 Chla h−1 (Fig. 3a). In the nif2 mutant strain, JE21, no hydrogen was produced by 7 h after induction (data not shown). For strains FD and PW253 (V76I) in an N2 atmosphere, H2 production was approximately 25% of the value measured under argon (Fig. 3a). Thus, in the presence of N2 about 25% of the electrons reduced protons and about 75% were likely devoted to reducing N2, whereas in the absence of N2 they were used solely for proton reduction. Strains PW357 (V75I) and PW350 (V75I, V76I), both with the V75I substitution that corresponds to the V70I substitution in A. vinelandii, produced about 90% as much H2 in an N2 atmosphere as in argon (Fig. 3a). The similar rates of H2 production under an argon atmosphere in the wild-type and V75I-substituted strains of A. variabilis suggest that this mutation largely, but not completely, blocks access of N2, but not protons, to the active site.