THIOLS AND NEURONAL NITRIC-OXIDE SYNTHASE - COMPLEX-FORMATION, COMPETITIVE-INHIBITION, AND ENZYME STABILIZATION

To elucidate how thiols affect neuronal nitric oxide synthase (nNOS) w e studied the binding of thiols to tetrahydrobiopterin (BH4)-free nNOS . Dithiothreitol (DTT), 2-mercaptoethanol, and L- and D-cysteine all b ound to the heme with K-d values varying from 0.16 mM for DTT to 31 mM for L-cysteine. DTT, 2-mercaptoethanol, and L-cysteine yielded absorb ance spectra with maxima at about 378 and 456 nm, indicative of bisthi olate complexes; the maximum at 426 nm with D-cysteine suggests bindin g of the neutral thiol. From the results with 2-mercaptoethanol we ded uced that in 2-mercaptoethanol-free, BH4-free nNOS the sixth heme liga nd is not a thiolate. DTT binding to nNOS containing one BH4 per dimer was biphasic. Apparently, the BH4-free subunit bound DTT with the sam e affinity as the BH4-free enzyme, whereas the BH4-containing subunit exhibited a >100-fold lower affinity, indicative of competition betwee n DTT and BH4 binding. Binding of DTT to the BH4-containing subunit wa s suppressed by L-arginine, whereas high-affinity binding was not affe cted, suggesting that L-arginine binds only to the BH4-containing subu nit. DTT competitively inhibited L-citrulline production by nNOS conta ining one BH4 per dimer (Ki approximate to 11 mM). Comparison of DTT b inding and inhibition suggests that the heme of the BH4-free subunit i s not involved in catalysis. Thermostability of nNOS was studied by pr eincubating the enzyme at various temperatures prior to activity deter mination. At nanomolar concentrations, nNOS was stable at 20 degrees C but rapidly deactivated at higher temperatures (t(1/2) approximate to 6 min at 37 degrees C). At micromolar concentrations, inactivation wa s 10 times slower. Absorbance and fluorescence measurements demonstrat e that inactivation was not accompanied by major structural changes. T he stabilization of nNOS by thiols was illustrated by the fact that om ission of 2-mercaptoethanol during preincubation for 10 min at 30 degr ees C led to an activity decrease of up to 90%.