Cysteine 184 of endothelial nitric oxide synthase is involved in heme coordination and catalytic activity.
Nitric oxide synthase catalyzes the formation of an important messenger molecule, nitric oxide (NO). It is a P450-type hemoprotein, containing a cysteine thiolate as its proximal heme ligand, but the exact cysteine residue involved in heme coordination has not been identified. To locate this specific cysteine, we altered three potential cysteine residues (Cys-99, Cys-184, and Cys-441) to alanine residues in human endothelial nitric oxide synthase (eNOS) by oligonucleotide-directed mutagenesis and expressed the wild-type and mutant eNOSs in COS-1 and the baculovirus expression system. Mutation of Cys-235 to alanine was included to serve as a control. Mutation of Cys-184 resulted in a complete loss of NOS catalytic activity and abrogation of the formation of carbon monoxide (CO)-heme ferrous complex, which was detected on CO difference spectra as a distinct peak centered on 444-446 nm, without reduction in the quantity of eNOS protein. Mutation of Cys-99 also resulted in a loss of catalytic activity but did not eliminate the 444-446 nm peak. C441A and C235A mutants displayed considerable NOS activity and retained the CO-heme peak on CO-ferrous difference spectra. These results indicate that the cysteine 184 of human eNOS is most likely the proximal heme ligand.
Chen
P F
PF
Vascular Biology Research Center, University of Texas Health Science Center, Houston 77030.
Tsai
A L
AL
Wu
K K
KK
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine; D004730:Endothelium, Vascular / Q000201:enzymology*; D006418:Heme / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation; D019001:Nitric Oxide Synthase
D008024:Ligands; D006418:Heme; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D003545:Cysteine