Effects of chemical modifications of heme on kinetics of carbon monoxide binding to free heme.

The rates of carbon monoxide recombination to six different kinds of chemically modified heme with various substituents at positions 2 and 4 have been studied in the proteinfree state (free heme) by the laser flash photolysis method in a mixture of ethylene glycol and 0.02 N NaOH (80:20, V/V) (80% ethylene glycol). The carbon monoxide combination rate constants to the various free hemes obtained in 80% ethylene glycol at 22” were 1.4, 2.1, 2.1, 3.7, 4.5, and 6.4 x 10’ M-’ s-’ for 2,4-diformyl-, spirographis (2-formyl-4-vinyl-), isospirographis (2-vinyl-4-formyl-), proto-(2,4-divinyl-), deutero-(2,4-dihydrogen-), and meso-(2,4-diethyl-1, hemes, respectively. This order of increase in carbon monoxide combination rate constants for these hemes correlates exactly with decrease in electron attractivity of heme side chains (i.e. increase in pK,, basicity of nitrogen base of porphyrin) and is completely opposite to that obtained for carbon monoxide binding to these hemes reconstituted with apomyoglobin (Sono, M., Smith, P. D., McCray, J. A., and Asakura, T. (1976) J. Biol. Chem. 251, 1418-1426). Contrary to the results for myoglobin, the two isomers of monoformyl-monovinylheme exhibited similar optical properties and the same combination rate constant indicating that the differences in the optical and kinetic results observed in myoglobin are due to different interactions of these isomeric hemes with protein. Deuteroheme combined with carbon monoxide at a rate between those of mesoand protohemes, as predicted from pK, values of porphyrins, also indicating that the anomalous behavior of deuteromyoglobin was due to the influence of protein in the heme pocket. The comparison of the kinetic difference spectrum with equilibrium difference spectrum suggests that the photodissociated carboxyheme has a different coordination structure (pyramidal) and optical property from the reduced heme (octahedral) observed in an equilibrium condition in 80% ethylene