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In these studies, we substitute electron-withdrawing (diacetyl) or -donating (diethyl) groups at the 2- and 4-positions of the heme in sperm whale Mb and HRP, and examine the structural and biochemical consequences. X-ray absorption spectroscopy shows that increased electron density 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 constrained by a H-bonding network, and axial effects are manifested entirely at the distal site. Conversely, in Mb, where the proximal ligand is less constrained, axial effects are seen at the proximal side. In HRP, electron density at the heme iron depends linearly on pK3, a measure of the basicity of the porphyrin pyrrole nitrogens [Yamada, H., Makino, R., & Yamazaki, I. (1975) Arch. Biochem. Biophys. 169, 344-353]. Using diethyl substitution (pK3 = 5.8) and diacetyl substitution (pK3 = 3.3) in HRP and Mb, we measured the one-electron reduction potentials (E(O)') of HRP compounds I and II and ferryl Mb. Compound I showed a decreased E(O)' with increasing electron density at the heme (pK3), similar to E(O)' of ferric HRP. E(O)' of HRP compound II and ferryl Mb showed an opposite dependence. This behavior of E(O)', while initially surprising, can be explained by the apparent net positive charge on the iron porphyrin in each oxidation state of the hemoproteins.

Upon the addition of hydrogen peroxide or ethyl hydroperoxide to sperm whale metmyoglobin (MbIII) in the presence of ethanol, MbIII was converted to oxymyoglobin (MbIIO2) under aerobic conditions and carboxymyoglobin (MbIICO) under CO-saturated conditions. MbIIO2 and MbIICO were also formed when ethanol was added to ferryl myoglobin (MbIV) which had been formed from the reaction of MbIII with hydrogen peroxide. From the stoichiometry, the primary reaction was formulated as follows. MbIV + ethanol----MbII + acetaldehyde The reaction was optimal at pH 7.0-7.5. Sperm whale ferryl myoglobin was reduced less effectively by methanol and n-butanol, but not at all by sec- and tert-butanols. The reduction of ferryl hemoproteins by ethanol was slower with horse heart myoglobin and was not observed with bovine hemoglobin or horseradish peroxidase.

It was found from spectrophotometric titration and proton balance measurement that the pKa value of a heme-linked protonation group of horseradish ferro-peroxidase C (donor:H2O2 oxidoreductase, EC 1.11.1.7) shifted from 7.25 to 8.25 upon combination with CO. The spectrophotometric titration experiment with myoglobin also revealed the presence of a heme-linked protonation group, the pKa value being 5.57 in myoglobin and 5.67 in the CO-myoglobin complex. It was concluded that the distinct shift of the pKa value in the case of peroxidase was attributable to the presence of a hydrogen bond between the sixth ligand and the distal base. The difference in the strength of such hydrogen bonding between peroxidase and myoglobin was discussed.

The heme-linked protonation of a ferrous horseradish peroxidase is assigned to a distal amino acid residue. The conclusion is drawn from analyses of reactions that involve the protonation. Unfortunately it is difficult to apply it to the myoglobin case because no reaction has been found which is coupled with the protonation of the distal histidine. It is therefore of special interest to note that the pK value of 5.7 has been assigned to the distal histidine of metmyoglobin from binding kinetics with ligands20). It is well known that the reaction with hydrogen peroxide is quite different for the two types of hemoproteins. The distal base may be associated with the stabilization of the primary compound with hydrogen peroxide and also another amino acid residue may serve as a nucleophile for the stabilization of the pi-cation radical of porphyrin in the case if peroxidases. Numerous papers have dealt with heme substitution, heme linked protonation and reactions of hemoproteins related to the present subject. In this short paper, however, the discussion has mostly centered around the data obtained recently in our laboratory.

Two heme-linked groups of horseradish peroxidases, characterized by pKa = 5.8 (isoenzyme A) and 7.3 (isoenzyme C), are believed to be distal amino acid residues and, judging by their acid-base properties in various derivatives, influence the functions of these isoenzymes. In contrast, in myoglobin ionization of the distal histidine does not influence its reactivity. Accordingly, we conclude that the interaction of the distal base with a 6th ligand is weak in myoglobin but very strong in peroxidases.

The stopped flow and flash photolysis methods were applied for the kinetic study of the reaction of carbon monoxide with myoglobin in solution, in an amorphous state, and in crystals. From the flash photolysis data, the reactivity of myoglobin was concluded to be essentially the same in all three states. When the reaction was started with a stopped flow apparatus, the rate became slower as the state of myoglobin was changed from solution to amorphous precipitate, and to crystals. The bigger the crystals, the slower the reaction became. Therefore, the change of the rate could be explained in terms of a diffusion layer formed on the crystals. In the reaction of crystalline myoglobin, the effective concentration of CO was increased locally by about 20 microM after flash photolysis and approached the bulk concentration during the reaction. In contrast, in the reaction of amorphous precipitate of myoglobin, such an increase in the CO concentration was apparently dispersed homogeneously just after flash photolysis.