Heme-linked ionization

Lignin peroxidase, secreted by the white-rot fungus Phanerochaete chrysosporium in response to nutrient deprivation, catalyzes the H202-dependent oxidation of non-phenolic aromatic substrates. The present report summarizes the kinetic and structural characteristics of lignin peroxidase isozymes. Our results indicate that the active site of lignin peroxidase is more electron deficient than other peroxidases. As a result, the redox potential of the heme active site is higher, the heme active site is more reactive and the oxycomplex is more stable than that of other peroxidases. Also discussed is the heme-linked ionization of lignin peroxidase. 

Studies on the effect of pH on peroxidase catalysis, or the heme-linked ionization, have provided much information on peroxidase catalysis and the active site structure. Heme-linked ionization has been observed in kinetic, electrochemical, absorption spectroscopic, proton balance, and Raman spectroscopic studies. Kinetic studies show that compound I formation is base-catalyzed (72). The pKa values are in the range of 3 to 6. The reactions of compounds I and II with substrates are also pH-dependent with pKa values in a similar range (72). Ligand binding (e.g. CO, O2 or halide ions) to ferrous and ferric peroxidases is also pH-dependent. A wide range of pKa values has been reported (72). The redox potentials of Fe3+/Fe2+ couples for peroxidases measured so far are all affected by pH. The pKa values are between 6 and 8, indicative of an imidazole group of a histidine residue (6, 31-33),... 

There is some experimental evidence showing the effects of heme-linked ionization on lignin peroxidase. The redox potential of the Fe +/Fe2+ couple of lignin and Mn-dependent peroxidases is pH-dependent, as is the absorption spectrum of the ferrous form of the lignin and Mn-dependent peroxidases (6). The pKa values determined from both experiments were 6.6. Detailed studies were performed studying O2 binding to the ferrous lignin peroxidase (79). The pKa for the ionization was measured at different... 

Hashimoto S, Tatsuno Y, Kitagawa T (1986) Resonance Raman evidence for oxygen exchange between the FeIV=0 Heme and bulk water during enzymic catalysis of horseradish peroxidase and its relation with the heme-linked ionization. Proc Natl Acad Sci USA 83 2417-2421... 

Sitter AJ, Reczek CM, Temer J (1985) Heme-linked ionization of horseradish peroxidase compound II monitored by the resonance Raman Fe (IV) = O stretching vibration. J Biol Chem 260 7515-7522... 

Evidence that there is protein on both sides of the hemes in hemc lobin has been at hand for tw nty years. There are two important heme-linked acid groups in the molecule, one of which is made more strongly acidic and the oilier less stroi ly acidic on oxygenation of the associated heme. It was suggested by Conant (14) that both of these acid g roups are imidazole grronps of histidine residues, and strong evidence for this assumption, from heats of ionization. 

The state of ionization of the imidazole residue linked to hemoglobin iron has no hitherto observed influence on the light absorption of hemi-, hemo-, or oxyhemoglobin. It seems possible, however, that very careful spectrophotometric determinations might reveal slight differences corresponding to the pK values of the heme-linked imidazole groups. 

The oxygen affinity of hemoglobin varies with the pH of the medium. This is the Bohr effect, and arises from the effect of pH on the interaction between the heme and the ionizable groups of the protein. It appears that the Bohr effect is linked to the presence of a second imidazole group (the distal imidazole group), which is involved in uptake of dioxygen in some indirect way. 

For example, Hayashi et al. (73) recently reported an enhancement of the peroxidase activity of Mb by modification of two heme-propionate side chains. They prepared an artificial hemin having two benzene moieties linked at each terminal carboxylate of the heme-propionates in protoheme IX, as shown in Fig. 18. The modified hemin was then inserted into the horse heart apoMb to yield a reconstituted Mb. The characterization of the reconstituted Mb was carried out by ultraviolet-visible (UV-vis), NMR, and electronspray ionization-mass spectrometry (ESI-MS). Particularly, the UV-vis spectrum of the reconstituted Mb is comparable with that observed for the native Mb, suggesting that the artificial hemin is located in the normal position of the heme pocket. 

We may combine this result with that given in the last section to obtain the heat of reaction AHo (per heme) of hemoglobin and carbon monoxide as it occurs in solution, neglecting the heat of ionization of the carbon-monoxide-linked acid groups, in the following way ... 

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