Expression of human inducible nitric oxide synthase in a tetrahydrobiopterin (H₄B)-deficient cell line: H₄B promotes assembly of enzyme subunits into an active dimer

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically active only in dimeric form. Assembly of its purified subunits into a dimer requires H₄B. To understand the structure-activity relationships of human iNOS, we constitutively expressed recombinant human iNOS in NIH 3T3 cells by using a retroviral vector. These cells are deficient in de novo H₄B biosynthesis and the role of H₄B in the expression and assembly of active iNOS in an intact cell system could be studied. In the absence of added H₄B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H₄B or the H₄B precursor sepiapterin generated NO (74.1 and 63.3 nmol of nitrite per 10⁶ cells per 24 h, respectively). NO synthesis correlated with an increase in intracellular H₄B but no increase in iNOS protein. Instead, an increased percentage of dimeric iNOS was observed, rising from 20% in cytosols from unsupplemented cells to 66% in H₄B-supplemented cell cytosols. In all cases, only dimeric iNnS displayed catalytic activity. Cytosols prepared from H₄B- deficient cells exhibited little iNOS activity but acquired activity during a 60- to 120-min incubation with H₄B, reaching final activities of 60-72 pmol of citrulline per mg of protein per min. Reconstitution of cytosolic NO synthesis activity was associated with conversion of monomers into dimeric iNnS during the incubation. Thus, human iNOS subunits dimerize to form an active enzyme, and H₄B plays a critical role in promoting dimerization in intact cells. This reveals a posttranslational mechanism by which intracellular H₄B can regulate iNOS expression.