Genes encoding for proteins with high sequence homology to the heme-containing, oxygenase domain of mammalian nitric oxide synthase (NOS) have been identified in a number of bacteria. Many of these species of bacteria do not contain the genes that encode for the synthetic machinery to produce tetrahydrobiopterin (H(4)B), a cofactor of NOS required for NO synthesis. These bacteria have the genes for the synthesis of tetrahydrofolate (H(4)F) which contains the redox-active pteridine ring of H(4)B. These observations led us to investigate whether H(4)F could be used for the synthesis of NO by NOS-like enzymes from bacteria that cannot make H(4)B. The NOS gene from one such bacterium, Deinococcus radiodurans, was cloned and expressed (deiNOS) in Escherichia coli and then purified and characterized. The K(D) of deiNOS for the NOS substrate arginine (0.9 +/- 0.1 mM) drops by over 2 orders of magnitude in the presence of H(4)F (7.4 +/- 0.1 microM). Further, NO is synthesized from the NOS substrate N-hydroxy-l-arginine (NHA) by deiNOS in the presence of H(4)F. Stopped-flow spectroscopic data reveal that H(4)F accelerates the rate of decay of the ferrous-oxy/ferric-superoxo species in substrate turnover. These data strongly suggest that H(4)F may be used by D. radiodurans to replace H(4)B as a redox-active cofactor for nitric oxide synthesis.