ENDOTHELIAL NITRIC-OXIDE SYNTHASE - MODULATIONS OF THE DISTAL HEME SITE PRODUCED BY PROGRESSIVE N-TERMINAL DELETIONS

cDNAs coding for bovine endothelial nitric oxide synthase (eNOS) with N-terminal deletions of 52, 91, and 105 amino acids were constructed, and the proteins were expressed in Escherichia coli and purified by af finity chromatography. All three truncated proteins bind heme and exhi bit the ferrous-CO absorption maximum at 444 nm characteristic of thio late heme ligation. Deletion of the first 52 amino acids yields a full y active dimeric protein with the same spectroscopic properties as the wild-type. The myristoylation, palmitoylation, and polyproline domain s of the enzyme located in the deleted region are therefore not requir ed for full catalytic activity. The Delta(91) and Delta(105) proteins, which exhibit altered dimerization equilibria, retain 20 and 12%, res pectively, of the maximal activity, Resonance Raman and UV-vis spectro scopy indicate that, in the absence of tetrahydrobiopterin (H4B) and L -Arg, the wild-type and Delta(52) proteins are predominantly five coor dinate high spin, whereas the Delta(91) and Delta(105) proteins are si x coordinate low spin. The Delta(91) and Delta(105) mutants bind H4B, as Indicated by a concomitant decrease in the low-spin component of th e UV-vis spectrum, but the binding of L-Arg is extremely slow (similar to 15 min), Dithiothreitol readily coordinates as the sixth iron liga nd in the Delta(91) and Delta(105) mutants but not in the Delta(52) or wild-type proteins, The dithiothreitol can be completely displaced by L-Arg but not by H4B. Resonance Raman comparison of wild-type eNOS an d nNOS confirms that, in the absence of H4B and L-Arg, eNOS is primari ly high spin whereas nNOS is predominantly six coordinate, low spin. T he results indicate that Cys-101 is not critical for the binding of H4 B and imply that some of the protein residues involved in dimer format ion and in preservation of active site integrity are located, probably at the monomer-monomer interface, in the N-terminal end of the protei n.