Differential effects of pH and inositol hexaphosphate on the spectroscopic properties of the alpha and beta subunits in methemoglobins M Milwaukee and A

Abstract

The effect of pH and inositol hexaphosphate on the electron spin resonance spectra of the α-hemes ( g = 6.0) and the β-hemes ( g = 6.7) has been measured in methemoglobin M Milwaukee and compared with that of methemoglobin A ( g = 6.0). The β-hemes are found to be comparatively insensitive to both effectors while the α-hemes behave in a manner similar to the heme groups of methemoglobin A. Binding of inositol hexaphosphate enhances the high spin ESR signal of the α-hemes in both methemoglobins. Comparison of the optical properties of methemoglobins A and M Milwaukee over the pH range from 5.0 to 8.1 shows that inositol hexaphosphate has a differential effect on the subunit types in these two methemoglobins. At low pH the spectral changes observed upon inositol hexaphosphate binding arise primarily from the β-hemes, while at neutral and alkaline pH these changes arise from both subunit types. The β-heme spectral changes appear to be pH independent while those arising from the α-hemes are strongly pH dependent. It is concluded that it is the hydroxymet form of the α-hemes which undergoes spectral change upon inositol hexaphosphate binding to the β-subunits. In methemoglobin A the spin state and paramagnetic susceptibility increase only in the neutral and alkaline pH ranges upon inositol hexaphosphate binding (Gupta, R.K. and Mildvan, R.S. (1975) J. Biol. Chem. 250, 246; Perutz, M.F., Sanders, J.K.M., Chenery, D.H., Noble, R.W., Penelly, R.R., Fung, L.W.-M., Ho, C., Giannini, I., Porschke, D. and Winkler, H. (1978) Biochemistry 17, 3640). Therefore the hydroxymet form of the α-hemes which is responsible for the observed spectral changes must also be responsible for these increases in the magnetic properties of methemoglobin A. Inositol hexaphosphate can bind to methemoglobin at alkaline pH if the β-hemes are in the high spin form.