Endothelial Nitric Oxide Synthase: Modulations of the Distal Heme Site Produced by Progressive N-Terminal Deletions

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Abstract
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 affinity chromatography. All three truncated proteins bind heme and exhibit the ferrous-CO absorption maximum at 444 nm characteristic of thiolate heme ligation. Deletion of the first 52 amino acids yields a fully active dimeric protein with the same spectroscopic properties as the wild-type. The myristoylation, palmitoylation, and polyproline domains of the enzyme located in the deleted region are therefore not required for full catalytic activity. The Δ91 and Δ105 proteins, which exhibit altered dimerization equilibria, retain 20 and 12%, respectively, of the maximal activity. Resonance Raman and UV−vis spectroscopy indicate that, in the absence of tetrahydrobiopterin (H4B) and l-Arg, the wild-type and Δ52 proteins are predominantly five coordinate high spin, whereas the Δ91 and Δ105 proteins are six coordinate low spin. The Δ91 and Δ105 mutants bind H4B, as indicated by a concomitant decrease in the low-spin component of the UV−vis spectrum, but the binding of l-Arg is extremely slow (∼15 min). Dithiothreitol readily coordinates as the sixth iron ligand in the Δ91 and Δ105 mutants but not in the Δ52 or wild-type proteins. The dithiothreitol can be completely displaced by l-Arg but not by H4B. Resonance Raman comparison of wild-type eNOS and nNOS confirms that, in the absence of H4B and l-Arg, eNOS is primarily high spin whereas nNOS is predominantly six coordinate, low spin. The results indicate that Cys-101 is not critical for the binding of H4B and imply that some of the protein residues involved in dimer formation and in preservation of active site integrity are located, probably at the monomer−monomer interface, in the N-terminal end of the protein.