Synthesis of nitric oxide by the NOS-like protein from Deinococcus radiodurans: A direct role for tetrahydrofolate

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 (HB), a cofactor of NOS required for NO synthesis. These bacteria have the genes for the synthesis of tetrahydrofolate (HF) which contains the redox-active pteridine ring of H B. These observations led us to investigate whether HF could be used for the synthesis of NO by NOS-like enzymes from bacteria that cannot make HB. The NOS gene from one such bacterium, Deinococcus radiodurans, was cloned and expressed (deiNOS) in Escherichia coli and then purified and characterized. The K of deiNOS for the NOS substrate arginine (0.9 ± 0.1 mM) drops by over 2 orders of magnitude in the presence of HF (7.4 ± 0.1 μM). Further, NO is synthesized from the NOS substrate N-hydroxy-L-arginine (NHA) by deiNOS in the presence of HF. Stopped-flow spectroscopic data reveal that HF accelerates the rate of decay of the ferrousoxy/ferric-superoxo species in substrate turnover. These data strongly suggest that HF may be used by D. radiodurans to replace HB as a redox-active cofactor for nitric oxide synthesis. © 2009 American Chemical Society.