CMldmyuOnas rihardii cells, growning photoautotropbical under air, excreted to the culture medium much higher amounts of N02 and NH4 I under ble than under red fight. Under shmilar conditions, but with N02-as the only nitrogen source, the cells consumed NO2 -and excreted NH4, at shilar rates under blue and red light. In the presence of N03-and air with 2% CO2 (v/v) incorporate al the pbotogenerated NH4 +. Because these cells should have high levels of reducing power, they might use N03-or, in its absence, NO,-as terminal electron acceptors. The excretion of the products of N02-and NH4, to the medium may provide a mechanism to control reductant level in the cells. Blue lght is suggested as an important regulatory factor of this photorespiratory conmption of N03-and possibly of the whole nitrogen metabolism in green algae.Nitrate assimilation in green algae and higher plants is a basic metabolic process because it uses more than 20o of the reducing power generated by their photosynthetic apparatus (11). Among the different steps involved in this metabolic pathway, reduction of NO3 to N02 catalyzed by nitrate reductase has become particularly relevant due to its regulatory features on nitrogen metabolism (5,12,36).NADH-nitrate reductase from green algae is a multimeric enzyme of high mol wt with several electron transport components such as flavin adenine dinucleotide, protoheme b557, and molybdenum (8). Recently, it has been reported that molybdenum is held in a special cofactor that contains an unidentified pterin (13).
Molybdenum is absolutely required for the nitrate-reducing activity of the nicotinamide adenine dinucleotide nitrate reductase complex isolated from Chlorella fusca. The The assimilatory nitrate-reducing system of the green alga Chlorella has been thoroughly characterized in recent years (13-16, 21, 23, 24, 28, 29) and has been shown to be similar to that of higher plants (6,9 (6,8,9,17). However, although molybdenum was early identified by Nicholas and Nason (19) as the metal prosthetic group of nitrate reductase from soybean leaves, workers in the field (3, 6, 14) have until recently been unable to find evidence for either its presence or its function as an electron carrier in nitrate reductase preparations purified from a variety of green plants species. Hewitt and coworkers (1, 2, 9) have reported that, in molybdenum-deficient plants grown in the presence of nitrate, molybdenum is required for the synthesis of nitrate reductase and have suggested that it is involved in the induction process and acts not merely as the constituent metal. Heimer et al. (7) and Wray and Filner (27) have recently studied the effect of tungstate as a competitive inhibitor of molybdate on nitrate assimilation in higher plant tissues and have described the structural and functional relationships of enzyme activities induced by nitrate in barley. Their results support the idea that the nitrate reductase complex is formed in the presence of tungstate but is functional only with respect to NADH-diaphorase. By adding radioactive 9Mo (as molybdate) to a culture of Chlorella cells at the moment derepression of the enzymes of the nitrate-reducing system was initiated by removal of ammonia from the medium, we have recently been able to demonstrate that the metal becomes incorporated into nitrate reductase and remains associated with it during purification (3). Furthermore, we showed that after a mild heat treatment of the enzyme exogenous molybdate chemically reduced by hydrosulfite can be used as electron donor for the enzymatic reduction of nitrate (3).The present work is intended to clarify the role played by molybdenum in nitrate reduction by Chlorella. MATERIALS AND METHODSChlorella fusca Shihira et Kraus (pyrenoidosa, strain 211-15 from Pringsheim's culture collection at Gottingen) was grown autotrophically as described previously (29). When ammonia or ammonium nitrate replaced nitrate as the source of nitrogen, the molarity of each compound was also maintained at 8 mM. In the experiments where ammonium ions were involved, the culture media were buffered with 20 mm sodium phosphate, pH 7.5. In the experiments involving the effect of metals, molybdate was omitted from the standard nutrient solution and either sodium molybdate or sodium tungstate was added as indicated. The algae used for inoculation were grown on nitrate media lacking added molybdate in order to produce a low molybdenum inoculum. Growth was determined by meas-294 www.plantphysiol.org on March 22, 2019 -Published by Downloaded from
Chlamydomonas reinhardii cells, after a period of dark anaerobic adaptation, evolve H2 not only in the dark but also in the light. Our results show that high irradiances impair prolonged H2 evolution, while under low irradiances or darkness H2 evolution proceeds for more than 50 hours. NO3-and NO2-suppress H2 evolution both in the dark or under low irradiance. Apparently the cells prefer these oxidized nitrogen sources to protons as electron acceptors, since both N03-and NOTbecome reduced to NHW+, which is excreted to the culture medium in high amounts. H2 evolution started once these oxidized anions were largely depleted from the medium. Moreover, H2 evolution was consistently associated with NH4I excretion even if NH4' was already present in high amounts in the medium. This observation indicates that the cells utilize not only their carbohydrate but also their protein reserves as sources of reducing power for H2 evolution. This conclusion was supported by the observation that when nitrogen-starved cells were made anaerobic in a nitrogen-free medium, they not only evolved H2 at very high rates but excreted concomitantly NH4W up to concentrations in the millimolar range.After some controversy following the pioneering work of Gaffron and Rubin (1 1) on H2 evolution by green algae, it is now widely accepted that two different pathways exist in these organisms for H2 photoproduction with either water or carbohydrates as electron donors (1). Direct coupling between oxygenic photosynthetic activity and the H2 evolving system has been demonstrated to take place preferentially during initial periods of light exposure (7,12,22). Alternatively, simultaneous evolution of CO2 and H2 have been observed both in the dark (15) and in a light-dependent process involving solely PSI (18). In these two latter cases, cellular carbon reserves provide ultimately the reducing equivalents for H2 evolution, with H+ acting as final electron acceptors. Since H2 evolution implies such a simple redox reaction, it should provide an effective pathway for the disposal of excess internal reducing power, especially under low 02 tensions (18,26). A similar relief valve operates in anaerobic bacteria (1).In photosynthetic eukaryotes, the reductive utilization ofNO3-is carried out by two different enzymes: NAD(P)H-nitrate reductase which reduces N03 to N02 , and reduced ferredoxin-nitrite reductase which reduces N02-to NH4' (14 to the medium, most probably to unload excess photosynthetically generated reducing power (2, 3). Moreover, it was found that in this organism inorganic nitrogen metabolism was modulated by blue light (4).We report here the effect ofthe physiological electron acceptors N03 and N02 on H2 production by anaerobically adapted cells of C. reinhardii. When either of these oxidized nitrogen compounds were present, H2 production was suppressed, while, depending on the oxidized nitrogen source, NO2-and/or NH4' were released into the medium. However, with NH4' as the only nitrogen source, H2 evolution was enhanced. Furthermo...
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