We have investigated the cleavage of the bond between the heme iron and the proximal heme ligand, His388, in ferrous ovine prostaglandin endoperoxide H synthase-l (oPGHS-1) on nitric oxide (NO) binding by using resonance Raman spectroscopy. The Fe-NO and N-O vibrations are observed at 526 and 1667cm(-1) respectively, which is indicative of a five-coordinated, heme-NO complex. We also observed NO photolysis under low-power, continuous wave conditions, which reflects nongeminate rebinding of NO to the heme cofactor on the millisecond time scale. Furthermore, we attempted to make a six-coordinated heme-NO complex by adding imidazole (Im) to ferrous oPGHS-1-NO. However, ferrous oPGHS-1-NO remained five-coordinated even in the presence of 500 mM Im. The heme-NO complex remains bound in the heme pocket after Fe-His bond cleavage, and subsequent protein backbone movement is relatively small. Finally, we compared the structures of several heme proteins that form either five- or six-coordinated complexes with NO. Inspection of the heme-proximal His geometry did not reveal any common structural parameter that may be critical for Fe-His bond cleavage. However, the Fe-His stretching frequency [v(Fe-His)] shows a good correlation with Fe-His bond cleavage and is modulated by hydrogen bonding to the Na proton of the His. We propose that the strength of this hydrogen bond is the primary factor determining Fe-His bond cleavage: a strong hydrogen bond gives the His imidazolate character, which allows it to compensate for the NO trans ligand effect and to avoid Fe-His bond cleavage.