Reconstitution of Pterin-free Inducible Nitric-oxide Synthase*

Inducible nitric-oxide synthase (NOS) was expressed and purified in the absence of 6(R)-tetrahydro-l-biopterin (H₄B). Pterin-free NOS exhibits a Soret band (416–420 nm) characteristic of predominantly low spin heme and does not catalyze the formation of nitric oxide (·NO) (Rusche, K. M., Spiering, M. M., and Marletta, M. A. (1998) Biochemistry 37, 15503–15512). Reconstitution of pterin-free NOS with H₄B was monitored by a shift in the Soret band to 396–400 nm, the recovery of ·NO-forming activity, and the measurement of H₄B bound to the enzyme. As assessed by these properties, H₄B binding was not rapid and required the presence of a reduced thiol. Spectral changes and recovery of activity were incomplete in the absence of reduced thiol. Full reconstitution of holoenzyme activity and stoichiometric H₄B binding was achieved in the presence of 5 mm glutathione (GSH). Preincubation with GSH before the addition of H₄B decreased, whereas lower concentrations of GSH extended, the time required for reconstitution. Six protected cysteine residues in pterin-free NOS were identified by labeling of NOS with cysteine-directed reagents before and after reduction with GSH. Heme and metal content of pterin-free and H₄B-reconstituted NOS were also measured and were found to be independent of H₄B content. Additionally, pterin-free NOS was reconstituted with 6-methylpterin analogs, including redox-stable deazapterins. Reconstitution with the redox-stable pterin analogs was neither time- nor thiol-dependent. Apparent binding constants were determined for the 6-methyl- (50 μm) and 6-ethoxymethyl (200 μm) deazapterins. The redox-stable pterin analogs appear to bind to NOS in a different manner than H₄B.