Mycothiol (MSH) [1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-␣-D-glucopyranoside], isolated as the bimane derivative, was established to be the major thiol in Nocardia sp. strain NRRL 5646, a species most closely related to Nocardia brasiliensis strain DSM 43758 T . Thiol formation and detection of MSH-dependent formaldehyde dehydrogenase activity in cell extracts are relevant to the possible modulation of nitric oxide toxicity generated by strain NRRL 5646.Nocardia sp. strain NRRL 5646 contains the first nitric oxide (NO) synthase (NOS) enzyme system found in the bacterial world (3). This enzyme catalyzes a complex process by which arginine is oxidized to NO and citrulline. The roles of this novel Nocardia NOS system have been investigated (3,16,17,21,31). One facet of this work that became interesting was an evaluation of possible roles of thiols in modulating NO toxicity, serving as stable storage forms for NO, or in trafficking of NO (11,18,28,36). Evidence is gathered in this study that thiols are produced by Nocardia sp. strain NRRL 5646, and mycothiol (MSH) in particular may play an important role in the detoxification, if not the pathogenicity and regulation, of NO in NOS-containing bacteria (15,26,27). In this regard, Nocardia sp. strain NRRL 5646 represents a prototype counterpart model of glutathione (GSH) and S-nitrosoglutathione metabolism in eukaryotes (4, 6, 9-11, 13, 18, 19, 32).For analysis, Nocardia sp stain. NRRL 5646 was grown in soybean flour-glucose medium in a two-stage process (3,16,17,31). Cells harvested after 48 h from fully grown second-stage cultures were pelleted by centrifugation (8,000 ϫ g for 20 min) and subjected to French pressure homogenization (12,000 lb/ in 2 ) and subsequent centrifugation (100,000 ϫ g for 60 min). Extracts filtered through 0.22-m Millex-GV membranes were used for determinations of protein (1), thiols such as cysteine, GSH, and nitrosothiols (RSNO) formed when thiols react with NO produced by NOS in this organism.HgCl 2 converted RSNO in cell extracts to thiols plus nitrogen oxides that reacted with 2,3-diaminonaphthalene to form 2,3-naphthotriazole, a product determined by fluorescence spectrometry (12,20). Analyses showed 2.50 Ϯ 0.17 pmol NO 2 Ϫ /g (dry weight) of cells, reflective of RSNO concentrations present in cell extracts. Analysis of the same extracts by Ellman's method (7) using 5,5Ј-dithio-bis(2-nitrobenzoic acid) indicated that 3.04 Ϯ 0.02 mol thiol/g (dry weight) of cells were present in Nocardia cell extracts. Although individual RSNO could not be identified, Nocardia contained RSNO corresponding to about 1/1,000 of the total measured thiols.Specific Nocardia thiols were determined by N-(1-pyrenyl) maleimide (NPM) (29, 33) and monobromobimane (mBBr) (25) derivatization and subsequent high-performance liquid chromatography (HPLC) analysis. A modified NPM derivatization/HPLC method (33) showed that ␥-glutamyl-L-cysteine was the only cell extract component that corresponded to standards, and its concentration was estimated to be 0.14 ...