THE STRUCTURE-FUNCTION RELATIONSHIP AND REDUCTION POTENTIALS OF HIGH OXIDATION-STATES OF MYOGLOBIN AND PEROXIDASE

In these studies, we substitute electron-withdrawing (diacetyl) or -do nating (diethyl) groups at the 2- and 4-positions of the heme in sperm whale Mb and HRP, and examine the structural and biochemical conseque nces. X-ray absorption spectroscopy shows that increased electron dens ity at the heme results in an increased iron-pyrrole nitrogen average distance in both HRP and Mb, while decreased electron density results in shorter average distances. In HRP, the proximal ligand is constrain ed by a H-bonding network, and axial effects are manifested entirely a t the distal site. Conversely, in Mb, where the proximal ligand is les s constrained, axial effects are seen at the proximal side. In HRP, el ectron density at the heme iron depends linearly on pK(3), a measure o f the basicity of the porphyrin pyrrole nitrogens [Yamada, H., Makino, R., & Yamazaki, I. (1975) Arch. Biochem. Biophys. 169, 344-353]. Usin g diethyl substitution (pK(3) = 5.8) and diacetyl substitution (pK(3) = 3.3) in HRP and Mb, we measured the one-electron reduction potential s (E(0)') of HRP compounds I and II and ferryl Mb. Compound I showed a decreased E(0)' with increasing electron density at the heme (pK(3)), similar to E(0)' of ferric HRP. E(0)' of HRP compound II and ferryl M b showed an opposite dependence. This behavior of E(0)', while initial ly surprising, can be explained by the apparent net positive charge on the iron porphyrin in each oxidation state of the hemoproteins.