Abstract

How nitric oxide (NO) reacts with free thiols to form S-nitrosothiols (RSNOs) in vivo is a question of much debate. The effects of different chelators and added metals ions on NO and O 2 consumption, and S-nitrosocysteine (CysNO) formation in cysteine solutions were studied using amperometric (NO and O 2 electrodes) measurements and UV-vis absorption. The results support a free radical mechanism for CysNO formation that involves Cys · generation by reaction with · NO 2 at higher NO concentrations and with copper ions at lower NO concentrations. The mechanism of recombinant human brain calbindin D 28k (rHCaBP) S-nitrosation by CysNO or S-nitrosoglutathione (GSNO) was investigated in detail. First, an efficient rHCaBP expression and purification system was set up by subcloning the HCaBP gene into the pET15b vector and expressing the protein in BL21(DE3)pLysS host cells. A protein yield of >30 mg/L culture with >95% purity was obtained. UV-vis and circular dichroism absorption, intrinsic fluorescence and mass spectrometry measurements indicate that rHCaBP is S-nitrosated by CysNO. Of a total of five free cysteine residues 2.6 ± 0.05 and 4.4 ± 0.09 are S-nitrosated in Ca 2+ -loaded and Ca 2+ -free rHCaBP, respectively, as determined by the Saville assay. Intrinsic protein fluorescence was demonstrated to be a sensitive probe of protein S-nitrosation due to efficient Förster energy transfer (R 0 {598} 17 Å) between tryptophan donors and RSNO acceptors. Mass spectrometry and UV-vis absorption results support a mechanism for NO transfer from GSNO to rHCaBP that requires trace copper added as either Cu,Zn-superoxide dismutase (CuZnSOD) or CuSO 4 . CuZnSOD is an efficient catalyst of rHCaBP S-nitrosation via a mechanism involving reduction of its active-site Cu II by a number of the thiols in rHCaBP, giving rise to Cys · radicals. The Cu I ZnSOD formed catalyzes the reductive cleavage of GSNO to release NO, which reacts with the Cys · radical to yield the S-nitrosoprotein. Since exposure of rHCaBP to either CysNO or GSNO also leads to rapid S-thiolation, the mechanism of protein S-glutathiolation was investigated in detail. rHCaBP, human CuZnSOD (HCuZnSOD), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) were found to be S-glutathiolated in decomposed GSNO solutions. Fresh GSNO, reduced glutathione (GSH) or oxidized glutathione (GSSG) are not efficient S-glutathiolatiog agents for the proteins examined here. Based on analysis by mass spectrometry and UV-vis absorption, GSNO decomposition in the dark at room temperature yields glutathione disulfide S-oxide [GS(O)SG], glutathione disulfide S-dioxide (GSO2SG), and GSSG as products. A hydrolysis pathway yielding GSOH and nitroxyl HNO/NO - as intermediates is proposed based on inhibition of GSNO breakdown by dimedone, and nitroxyl scavenging by metmyoglobin. Cys111, Cys187, Cys149 and Cys34 were tentatively identified as the S-glutathiolation sites in HCuZnSOD, rHCaBP, GAPDH and BSA, respectively