Regulation of nitrogen fixation by the metabolism of carbon compounds

Nitrogen fixation in Rhodopseudomonas capsulata was subject to regulation by carbon metabolism. Citric acid cycle intermediates participated in this regulation, either directly or through accumulation of unknown derivatives of their metabolism. The carboxylic acids appeared to inhibit the A (or active) form of nitrogenase that predominates in nitrogen-starved cells. In exponentially growing cells in lactate-glutamate medium, where the R (or regulated) form of nitrogenase predominates, the citric acid cycle intermediates inhibited C2H, reduction activity by only 10-35%. Citrate and isocitrate were the most potent inhibitors, though full inhibition was not achieved before periods of one to several hours after their addition. The inhibitory effect of the 6-carbon carboxylic acids was not due to repression of enzyme synthesis, nor was it accompanied by changes in the adenylylation state of glutamine synthetase. Oxaloacetate, the immediate precursor of citrate, also caused inhibition of C2H3 reduction activity, but the inhibition was observed after 10-20 min. After an additional hour the inhibition was gradually relieved. It is suggested that oxaloacetate which penetrates the cell better than citrate and isocitrate, gives rise to a faster accumulation of the inhibiting compound. After a while cell metabolism is adjusted to prevent a high steady state concentration of oxaloacetate, and thereby accumulation of citrate or its derivatives is avoided. Inhibition by carboxylic acids of the A form of nitrogenase is of economical benefit for cell metabolism. Nitrogenase A is present when there is a carbon source in the growth medium but no nitrogen source. Under these conditions the cells metabolize carbon compounds via the citric acid cycle, and carbon sources and energy are wasted when the active form of nitrogenase is producing large amounts of hydrogen gas. However, since intermediates of the citric acid cycle are not removed for amino acid synthesis their accumulation causes inhibition of nitrogenase, thus preventing the waste.