BINDING OF CYANIDE AND THIOCYANATE TO MANGANESE RECONSTITUTED MYOGLOBIN AND FORMATION OF PEROXIDE COMPOUND - OPTICAL SPECTRAL, MULTINUCLEARNMR, AND KINETIC-STUDIES

The interaction of inorganic anions with manganese(III) protoporphyrin IX reconstituted myoglobin (Mn(III)Mb), coordination geometry around the metal ion in the reconstituted protein and the existence of higher oxidation states have been investigated by optical difference, multin uclear NMR and stopped flow techniques. The binding of (SCN)-N-15- and (CN)-C-15- to Mn(III)Mb was studied by optical difference spectroscop y and the observation of the spin lattice relaxation rate of the N-15 resonance. The values for dissociation constants for SCN- to Mn(III)Mb were found to vary from approximately 5 to approximately 94 mM in the pH range 5-10. The dissociation constant for the binding of CN- to Mn (III)Mb was measured by optical difference spectroscopy in competition with SCN-, which shows that the binding site for both CN- and SCN- is the same. The value of dissociation constant of CN- to Mn(III)Mb was found to be approximately 91 mM at pH = 9.2. These values show that th e binding of both CN- and SCN- is weak. The dissociation constant valu es measured from both optical and NMR method agree well. The pH depend ence of the line width and the dissociation constant shows that the bi nding involves a complex pH equilibrium among the amino acid residues in the region of the binding site; i.e. in this case more than one aci d half arrow right over half arrow left base equilibrium is operative. The distances measured for SCN- and CN- from Mn(III) center was found to be 6.7 and 6.2 angstrom. These observations are comparable to thos e for the Mn(III)HRP system, which suggest that the binding site is aw ay from the metal center. The measurements of water proton relaxivity at pH = 4.0 and 9.6 give the distances of the water proton from the Mn (III) center as 12.8 and 13.7 angstrom, respectively. This indicates t hat the water molecule is not present in the first coordination sphere of the Mn(III) center. Oxidation of Mn(III)Mb by excess H2O2 shows th at it forms a short-lived redox intermediate which has an optical spec trum similar to that of Mn(III)HRP compound I. The rate measurements b y stopped flow kinetics techniques show that the value of the apparent second-order rate constant of the formation of this higher oxidation state is low (2.5 M-1 s-1). The results bring out the importance of th e protein cavity around the metal in its peroxidase activity.