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J. Biol. Chem., Vol. 275, Issue 46, 35786-35791, November 17, 2000

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Role of Bound Zinc in Dimer Stabilization but Not Enzyme Activity of Neuronal Nitric-oxide Synthase^*

Benjamin Hemmens, Walter Goessler^§, Kurt Schmidt, and Bernd Mayer^¶

From the  Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, and ^§ Institut für Chemie, Karl-Franzens-Universität Graz, Universitätsplatz 1, Graz A-8010, Austria

Nitric-oxide synthases (NOS) are homodimeric proteins and can form an intersubunit Zn(4S) cluster. We have measured zinc bound to NOS purified from pig brain (0.6 mol/mol of NOS) and baculovirus-expressed rat neuronal NOS (nNOS) (0.49 ± 0.13 mol/mol of NOS), by on-line gel-filtration/inductively coupled plasma mass spectrometry. Cobalt, manganese, molybdenum, nickel, and vanadium were all undetectable. Baculovirus-expressed nNOS also bound up to 2.00 ± 0.58 mol of copper/mol of NOS. Diethylenetriaminepentaacetic acid (DTPA) reduced the bound zinc to 0.28 ± 0.07 and the copper to 0.97 ± 0.24 mol/mol of NOS. Desalting of samples into thiol-free buffer did not affect the zinc content but completely eliminated the bound copper (0.02 mol/mol of NOS). Most (75%) of the bound zinc was released from baculovirus-expressed rat nNOS by p-chloromercuriphenylsulfonic acid (PMPS). PMPS-treated nNOS was strongly (90 ± 5%) inactivated. To isolate functional effects of zinc release from other effects of PMPS, PMPS-substituted thiols were unblocked by excess reduced thiol in the presence of DTPA, which hindered reincorporation of zinc. The resulting enzyme contained 0.12 ± 0.05 mol of zinc but had a specific activity of 426 ± 46 nmol of citrulline.mg¹.min¹, corresponding to 93 ± 10% of non-PMPS-treated controls. PMPS also caused dissociation of nNOS dimers under native conditions, an effect that was blocked by the pteridine cofactor tetrahydrobiopterin (H₄biopterin). H₄biopterin did not affect zinc release. Even in the presence of H₄biopterin, PMPS prevented conversion of NOS dimers to an SDS-resistant form. We conclude that zinc binding is a prerequisite for formation of SDS-resistant NOS dimers but is not essential for catalysis.

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