No abstract

Nitrogenase and nitrogenase reductase disso-

Carbon monoxide inhibits reduction of dinitrogen (N2) by purified nitrogenase from Azotobacter vinelandii and Clostridium pasteurianum in a noncompetitive manner (Kii and Kis = 1.4 X 10(-4) and 4.5 X 10(-4) and 7 X 10(-4) atm and 14 X 10(-4) atm for the two enzymes, respectively). The onset of inhibition is within the turnover time of the enzyme, and CO does not affect the electron flux to the H2-evolving site. The kinetics of CO inhibition of N2 reduction are simple, but CO inhibition of acetylene reduction is complicated by substrate inhibition effects. When low-temperature (approximately 13 K) electron paramagnetic resonance (EPR) spectra of CO-inhibited nitrogenase are examined, it is found that low concentrations of CO ([CO] = [enzyme]) induce the appearance of a signal with g values near 2.1, 1.98, and 1.92 with t1/2 approximately 4 s, while higher concentrations of CO lead to the appearance of a signal with g values near 2.17, 2.1, and 2.05 with a similar time course. The MoFe proteins from Rhizobium japonicum and Rhodospirillum rubrum, reduced with Azotobacter Fe protein in the presence of CO, give similar results. Under conditions which promote the accumulation of H2 in the absence of CO, an additional EPR signal with g values near 2.1, 2.0, and 1.98 is observed. The use of Azotobacter nitogenase components enriched selectively with 57Fe or 95Mo, as well as the use of 13CO, permitted the assignment of the center(s) responsible for the induced signals. Only 57Fe, when present in the MoFe protein, yielded broadened EPR signals. It is suggested that the MoFe protein of nitrogenase contains one or more iron-sulfur clusters of the type found in the simple ferrodoxins. It is further proposed that the CO-induced signals arise from states of the MoFe protein in which CO inhibits electron flow to the N2-reducing site so that the iron-sulfur cluster achieves steady-state net charges of -1 (high CO complex) and -3 (low CO complex) in analogy to the normal paramagnetic states of high-potential iron-sulfur proteins and ferredoxins, respectively. The "no-CO" signal may be either an additional center or the N2-reducing site with H2 bound competitively.

A variety of gases, including H2, CO, N20, and NO, competitively inhibit N2 fixation.' Sch6llhorn and Burris2 and Dilworth3 independently observed that acetylene also inhibits N2 fixation, and Schollhorn and Burris2 reported that the inhibition is competitive. Dilworth3 observed that acetylene is reduced to ethylene by extracts from Clostridium pasteurianum.Methods and Materials.-The gases H2, N2 (high purity), and acetylene (purified grade) were commercial cylinder gases. Acetylene was freed from traces of acetone by condensing the acetone in a trap cooled with dry ice.Enzyme preparations: Cultures of Clostridium pasteurianum (strain W-5) were grown in a nitrogen-deficient medium with N2, harvested, dried in a rotary evaporator, and stored in evacuated tubes at -20°. Extracts were obtained by autolyzing the dried cells for 1 hr with shaking in 0.05 M cacodylate buffer pH 6.8 at 320 in an atmosphere of H2. The resulting suspension was centrifuged at 20,000 g for 25 min, and the supernatant was used.Azotobacter vinelandii (strain 0) was grown in aerated liquid cultures in 180-liter glass-lined fermentors, and the cells were stored as a frozen paste. N2-fixing extracts were prepared as described by Bulen, Burns, and LeComte4 and the supernatant of successive centrifugations at 35,000 g for 30 min and 144,000 g for 60 min was used.Experimental conditions: Experiments for inhibition of N2 fixation in C. pasteurianum were run in 20-ml rubber-stoppered serum bottles containing 1 ml of reaction mixture and the desired atmosphere; H2 was used as the electron donor. The mixture contained 5 /moles ATP, 50 Jmoles acetylphosphate, 2 emoles MgCl2, 50 /Amoles cacodylate buffer at pH 6.8, and enzyme preparation

Internal CO2 and O2 concentrations in Sedum praealtum DC. were determined by gas chromatography of 200-�l gas samples. Day-night monitoring showed that internal CO2 varied from a high of approximately 4000 �l/l during periods of daytime stomatal closure to a low of 270-280 �l/l during the dark period (stomata open). Internal O2 concentrations varied from a high of approximately 26 % at midday to a low of 20.8 % during the dark period. The calculated internal O2/CO2 ratio varied about 12-15-fold from 50-60 near midday to approximately 750 during the dark period (ratio in normal air is roughly 600). Day-night patterns of CO2 exchange and malic acid concentration were typical for a plant with crassulacean acid metabolism (CAM). Influx of CO2 during the late light period was inhibited by O2, but dark CO2 influx was O2-insensitive. Gas samples taken near midday from several CAM plants all showed elevated internal CO2 and O2 concentrations. Ratios of O2/CO2 in these plants ranged from 81 in Sedum praealtum to 285 in Hoya carnosa. The highest internal O2 concentration observed was 41.5% in Kalanchoe gastonis-bonnieri. The high CO2 concentration in leaves of CAM plants during daytime stomatal closure should provide a near- saturating level of this substrate for photosynthesis. In comparison to C3 plants, the relatively low O2/CO2 ratio in the CAM leaf during malic acid decarboxylation should be favourable for photosynthesis and unfavourable for O2 inhibition of photosynthesis.