SPECIFIC MODIFICATION OF STRUCTURE AND PROPERTY OF MYOGLOBIN BY THE FORMATION OF TETRAZOLYHISTIDINE-64(E7) - REACTION OF THE MODIFIED MYOGLOBIN WITH MOLECULAR-OXYGEN

Tetrazole-myoglobin (Tet-Mb), a site selectively modified myoglobin wi th tetrazole anion (-CN4-) covalently attached to the imidazole N(epsi lon) of the distal histidine 64(E7) (see Fig. 1; Kamiya, N., Shiro, Y. , Iwata, T., Iizuka, T., and Iwasaki, H. (1991) J. Am. Chem. Soc. 113, 1826-1829), exhibited unique properties in the reduction from ferric to ferrous states and in the reaction of its deoxy form with O2. The r edox potential of Tet-Mb is obtained to be -193 mV, which is much lowe r than that of unmodified (native) myoglobin (50 mV), possibly due to the electrostatic interaction between the heme iron and the tetrazole group. The ferrous deoxy form of Tet-Mb was rapidly oxidized to its fe rric form in the reaction with O2 at room temperature through an inter mediary formation of its oxy form and with the generation of O2-. The oxy form of Tet-Mb can be detected by the optical spectral measurement at -12-degrees-C, the rapid scan measurement at room temperature, and the electron spin resonance measurement of its cobalt-substituted der ivative (Tet-Mb(Co2+)) at 77 K. In the kinetic measurement of the O2 b inding reaction to Tet-Mb, its association and dissociation rate const ants in the bimolecular reaction were 6.1 x 10(7) M-1 s-1 and 2200 s-1 , respectively, showing that the tetrazole modification of His-64 extr emely accelerates its association and dissociation rates. Taken togeth er with the extremely fast autoxidation rate (53 h-1) obtained, these kinetic results suggested that the channel of O2 from the solvent regi on to the protein interior is open enough to pass the external ligand. The structure is discussed in relation to those of some genetic mutan ts. Taking these properties, we demonstrated that Tet-Mb can catalyze O2 consumption to generate O2-, coupled with the NADH-supported enzyma tic reduction system of cytochrome P-450cam under an aerobic condition .