EXPRESSION OF HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE IN A TETRAHYDROBIOPTERIN (H4B)-DEFICIENT CELL-LINE - H4B PROMOTES ASSEMBLY OF ENZYME SUBUNITS INTO AN ACTIVE

Murine inducible nitric oxide (NO) synthase (iNOS) is catalytically ac tive only in dimeric form. Assembly of its purified subunits into a di mer requires H4B. To understand the structure-activity relationships o f human iNOS, we constitutively expressed recombinant human iNOS in NI H 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 as sembly of active iNOS in an intact cell system could be studied, In th e absence of added H4B, NO synthesis by the cells was minimal, whereas cells grown with supplemental H4B or the H4B precursor sepiapterin ge nerated NO (74.1 and 63.3 nmol of nitrite per 10(6) cells per 24 h, re spectively). 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 unsupp lemented cells to 66% in H4B-supplemented cell cytosols. In all cases, only dimeric iNOS displayed catalytic activity. Cytosols prepared fro m H4B-deficient cells exhibited little iNOS activity but acquired acti vity during a 60- to 120-min incubation with H4B, reaching final activ ities of 60-72 pmol of citrulline per mg of protein per min, Reconstit ution of cytosolic NO synthesis activity was associated with conversio n of monomers into dimeric iNOS during the incubation. Thus, human iNO S subunits dimerize to form an active enzyme, and H4B plays a critical role in promoting dimerization in intact cells. This reveals a post-t ranslational mechanism by which intracellular H4B can regulate iNOS ex pression.