Ru(II)- and Re(I)-diimine wires bind to the oxygenase domain of inducible nitric oxide synthase (iNOSoxy). In the ruthenium wires, [Ru(L)2L'](2+), L' is a perfluorinated biphenyl bridge connecting 4,4'-dimethylbipyridine to a bulky hydrophobic group (adamantane, 1), a heme ligand (imidazole, 2), or F (3). 2 binds in the active site of the murine iNOSoxy truncation mutants Delta 65 and Delta 114, as demonstrated by a shift in the heme Soret from 422 to 426 nm. 1 and 3 also bind Delta 65 and Delta 114, as evidenced by biphasic luminescence decay kinetics. However, the heme absorption spectrum is not altered in the presence of 1 or 3, and Ru-wire binding is not affected by the presence of tetrahydrobiopterin or arginine. These data suggest that 1 and 3 may instead bind to the distal side of the enzyme at the hydrophobic surface patch thought to interact with the NOS reductase module. Complexes with properties similar to those of the Ru-diimine wires may provide an effective means of NOS inhibition by preventing electron transfer from the reductase module to the oxygenase domain. Rhenium-diimine wires, [Re(CO)(3)L1L1'](+), where L-1 is 4,7-dimethylphenanthroline and L-1' is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole to a distal imidazole (F(8)bp-im) (4) or F (F(9)bp) (5), also form complexes with Delta 114. Binding of 4 shifts the Delta 114 heme Soret to 426 nm, demonstrating that the terminal imidazole ligates the heme iron. Steady-state luminescence measurements establish that the 4:Delta 114 dissociation constant is 100 +/- 80 nM. Re-wire 5 binds Delta 114 with a K-d of 5 +/- 2 mu M, causing partial displacement of water from the heme iron. Our finding that both 4 and 5 bind in the NOS active site suggests novel designs for NOS inhibitors. Importantly, we have demonstrated the power of time-resolved FET measurements in the characterization of small molecule:protein interactions that otherwise would be difficult to observe.