TRANS EFFECTS ON CYSTEINE LIGATION IN THE PROXIMAL HIS93CYS VARIANT OF HORSE HEART MYOGLOBIN

Three variants of horse heart myoglobin (Mb) in which the proximal His 93 residue has been replaced with a Cys residue have been constructed and studied by NMR, EPR, and MCD spectroscopy to evaluate the contribu tions of proximal and distal residues to the coordination environment of the heme iron in these proteins. Although no experimental condition s were identified that allowed quantitative ligation of the cysteine r esidue to the heme iron in the His93Cys variant, all of the spectrosco pic evidence collected for the His93Cys/His64Ile and His93Cys/His64Val double variants supports the assignment of thiolate as the ligand to iron in the oxidized forms of these variants. The double metMb variant s exhibit Soret maxima that are considerably blue-shifted, H-1 NMR spe ctra with decreased mean methyl resonances, and EPR spectra with highl y rhombic g values. These spectroscopic data for the Fe(III) variants resemble the corresponding properties reported for ferricytochrome P-4 50. The decrease in the reduction potential of the double variants by 280 mV relative to wild-type protein is also consistent with the low m idpoint potential of cytochrome P-450. MCD spectroscopy of these varia nts confirms that the proximal cysteine residue is not bound in the re duced forms of these proteins and, in the case of the His93Cys variant , that the distal histidine is coordinated to the iron. Similar coordi nation environments were created in the ferrimyoglobin variants by cya nogen bromide modification, which resulted in cyanation of the sulfur atom and prevented the ligation of Cys93 to the heme iron. From these results it is apparent that simple mutagenenic modifications of the ac tive site of horse heart myoglobin can reproduce the characteristics o f ferricytochrome P-450, but that reproduction of the spectroscopic pr operties of ferrocytochrome P-450 will require more subtle modificatio ns.