J Biol Chem, Vol. 274, Issue 21, 14537-14540, May 21, 1999

COMMUNICATION
Zinc Content of Escherichia coli-expressed Constitutive Isoforms of Nitric-oxide Synthase
ENZYMATIC ACTIVITY AND EFFECT OF PTERIN

R. Timothy Miller, Pavel Martásek, C. S. Raman^¶, and Bettie Sue Siler Masters

From the  Department of Biochemistry, the University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7760 and the ^¶ Department of Molecular Biology and Biochemistry, the University of California, Irvine, California 92697-3900

Recently, we obtained x-ray crystallographic data showing the presence of a ZnS₄ center in the structure of Escherichia coli-expressed bovine endothelial nitric-oxide synthase (eNOS) and rat neuronal nitric-oxide synthase (nNOS). The zinc atom is coordinated by two CXXXXC motifs, one motif being contributed by each NOS monomer (cysteine 326 through cysteine 331 in rat nNOS). Mutation of the nNOS cysteine 331 to alanine (C331A) results in the loss of NO^· synthetic activity and also results in an inability to bind zinc efficiently. Although prolonged incubation of the C331A mutant of nNOS with high concentrations of L-arginine results in a catalytically active enzyme, zinc binding is not restored. In this study, we investigate the zinc stoichiometry in wild-type nNOS and eNOS, as well as in the C331A-mutated nNOS, using a chelation assay and electrothermal vaporization-inductively coupled plasma-mass spectrometry. The data reveal an approximate 2:1 stoichiometry of heme to zinc in (6R)-5,6,7,8-tetrahydro-L-biopterin-replete, wild-type nNOS and eNOS and show that the reactivated C331A mutant of nNOS has a limited ability to bind zinc. The present study substantiates that the zinc in NOS is structural rather than catalytic and is important for maintaining optimally functional, enzymatically active, constitutive NOSs.