Two types of conformers with distinct Fe-C-O configuration in the ferrous CO complex of horseradish peroxidase. Resonance Raman and infarared spectroscopic studies with native and deuteroheme-substituted enzymes.

T Uno,Y Nishimura,M Tsuboi,R Makino,T Iizuka,Y Ishimura

doi:10.1016/s0021-9258(18)61227-x

Abstract

The presence of at least two types of conformers in the ferrous CO complex of horseradish peroxidase has been demonstrated with the use of native and deuteroheme-substituted enzymes. Type I conformers, predominant in acidic pH, exhibited both an Fe-CO stretching and an Fe-C-O bending Raman line together with an infrared C-O stretch band below 1920 em-1. On the other hand, type II conformers, dominant species in alkaline pH, showed only an Fe-CO stretching Raman line with the C-O stretch above 1930 cm-1. They were interconvertible either by the changes in pH or by the binding of benzhydroxamate, a substrate for the enzyme. The pKa value for the pH-dependent interconversion of CO complex of deuteroheme-substituted enzyme was 8.3. These findings were interpreted to mean that the bound CO molecule in type I conformers was more tilted over the heme-plane than that in type II conformers. A steric hindrance by the bound substrate or the protonated form of a distal amino acid residue, presumably of histidine, is considered to be the cause for the isomerization. By summarizing present and previous data on the vibrational frequencies of heme-carbonyl complexes, we found that there are inverse-linear relationships between the square of Fe-CO and that of C-O stretching frequencies, while squares of Fe-CO stretching and Fe-C-O bending frequencies were linearly correlated with each other. Also found is that the dissociation rate constant of CO molecule from heme-carbonyl complexes is a linear function of the Fe-CO stretching frequency. The significance of these results is discussed.

Highlights

The presence of at least two typesof conformers in heme compounds [14,15,16]have been studied by resonance the ferrousCO complex of horseradish peroxidase has Raman and/or infraredspectroscopy for their Fe-CO stretchbeen demonstrated with the use of native and deute- ing, Fe-C-0 bending, and thebound C-0 stretching modes
The ers was more tilted over the heme-plane than that in results revealed that the CO complex was in equilibria of type I1 conformers
These findings indicate that the tran- Fig. 6B shows resonance Raman spectrain the region between sition between type I and type I1 conformers is caused by 700 and 450 cm” for the CO complex of native enzyme in the protonation of an amino acid residue at the distal side

Summary

MATERIALS AND METHODS

With slight modifications; the enzyme, dissolved in 35 mM sodium acetate buffer, pH 4.4, was applied to a column of CM-Sepharose CLCO complexes of hemoproteins such as oxygenases [1,2,3,4], 6B (1.5 X 10 cm, Pharmacia), which had been equilibrated with the oxidases [5,6,7], and oxygen carriers [8,9,10,11,12,13] as well as of model same buffer, and was eluted with a linear gradientof 0-30 mM NaC1. Apoenzyme of isozyme C was prepared ordination Fundsfor Promotion of Science and Technology from the accordingto themethod of Teale [22]. deuteroheme-substituted. Science and Technology Agency, Japan, andby grants from the Keio enzyme was prepared by reconstituting the apoenzyme with deut-. The costs of publication of this article were defrayed in eroheme by the method described previously [23]. This article must be cients of 102 m”’ cm” at 403 nm [24] and 107 mM” cm” at 393 hereby marked “aduertisement” in accordance with 18 U.S.C. Section nrn [23] were used t o calculate the concentration of native and. $ Present address: Dept of Pharmaceutical Science, Nagoya City The CO complex of the enzymes was prepared by adding a mini-

Present address

RESULTS

Findings

DISCUSSION