INFLUENCE OF AMINO-ACID REPLACEMENTS IN THE HEME POCKET ON THE ELECTRON-PARAMAGNETIC RESONANCE-SPECTRA AND ABSORPTION-SPECTRA OF NITROSYLHEMOGLOBINS M-IWATE, M-BOSTON, AND M-MILWAUKEE

In order to clarify the role of amino acids in the heme pocket, the electron paramagnetic resonance (EPR) spectra and the absorption spectra of the photolyzed form were obtained for nitrosylhemoglobins M in the fully reduced state. The EPR spectrum of the abnormal αNO subunit of nitrosylhemoglobin M Boston [His-E7(58)α→Tyr] exhibits readily resolved hyperfine splitting due to the 14N nucleus of bound NO and further superhyperfine splitting due to a trans axial ligand with I=1, which implies that His-F8 in the abnormal α subunit is coordinated to the heme iron. Addition of inositol hexaphosphate (IHP) to nitrosylhemoglobin M Boston produces strong triplet hyperfine structures at both the gy and gz regions of the spectrum, suggesting anomalous coordination of NO. The EPR spectra of the abnormal αNO subunit of nitrosylhemoglobin M Iwate [His-F8(87)α→Tyr] exhibit strong triplet hyperfines at both pH 6.5 and 9.0 regardless of the presence of IHP. NO of the abnormal α subunit presumably binds to the heme iron from the proximal side as in the carbonmonoxy form [Peisach, J., & Gersonde, K. (1977) Biochemistry 16, 2539-2545] and the bond between Fe and Nε(His-E7) must be weak, if present. The EPR spectrum of the abnormal βNO subunit in nitrosylhemoglobin M Milwaukee [Val-E11(67)β→Tyr] becomes similar to that of the αNO chain as a result of the mutation. Addition of IHP to nitrosylhemoglobin M Milwaukee produces the strong triplet hyperfine structure which is attributable to the αNO subunit. The absorption spectra of the photolyzed forms in the near-infrared regions were found to be sensitive to the amino acid replacement in the heme pocket. © 1979, American Chemical Society. All rights reserved.