Nitrogenase not only reduces atmospheric nitrogen to ammonia, but also reduces protons to hydrogen (H(2)). The nitrogenase system is the primary means of H(2) production under photosynthetic and nitrogen-limiting conditions in many photosynthetic bacteria, including Rhodospirillum rubrum. The efficiency of this biological H(2) production largely depends on the nitrogenase enzyme and the availability of ATP and electrons in the cell. Previous studies showed that blockage of the CO(2) fixation pathway in R. rubrum induced nitrogenase activity even in the presence of ammonium, presumably to remove excess reductant in the cell. We report here the re-characterization of cbbM mutants in R. rubrum to study the effect of Rubisco on H(2) production. Our newly constructed cbbM mutants grew poorly in malate medium under anaerobic conditions. However, the introduction of constitutively active NifA (NifA*), the transcriptional activator of the nitrogen fixation (nif) genes, allows cbbM mutants to dissipate the excess reductant through the nitrogenase system and improves their growth. Interestingly, we found that the deletion of cbbM alters the posttranslational regulation of nitrogenase activity, resulting in partially active nitrogenase in the presence of ammonium. The combination of mutations in nifA, draT and cbbM greatly increased H(2) production of R. rubrum, especially in the presence of excess of ammonium. Furthermore, these mutants are able to produce H(2) over a much longer time frame than the wild type, increasing the potential of these recombinant strains for the biological production of H(2). (C) 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.