EPR study of heme x NO complexes of ascorbic acid-reduced Pseudomonas cytochrome oxidase and corresponding model complexes.

B.B Muhoberac,D.C Wharton

doi:10.1016/s0021-9258(18)43515-6

Abstract

The EPR spectra of the NO complexes of frozen solutions of ascorbic acid-reduced cytochrome oxidase (nitrite reductase) purified from Pseudomonas aeruginosa, of its heme d1-depleted form, and of heme d1 in solutions containing various nitrogenous bases are quite similar to each other as well as to several heme (iron protoporphyrin IX)-containing proteins. The NO complexes of heme d1 (an iron-chlorin) in the presence of nitrogenous bases belong to spectral type C according to Kon's classification and, thus, the energy levels of the iron are closely related to thorse of heme complexes recorded under similar conditions. Comparison of these spectra with those of complexes of known structure suggests that both heme c and heme d1 are linked with Pseudomonas cytochrome oxidase by means of a nitrogenous ligand. The EPR spectrum of the NO complex of the native enzyme exhibits a lack of resolution of the high field (gy) resonance which can be characterized in terms of a spectral contribution from both the heme c and heme d1 moieties. The similarity between the EPR spectra of the NO complexes of horse heart cytochrome c and the heme d1-depleted Pseudomonas cytochrome oxidase before and after interaction with urea suggests structural similarities involving the heme irons. The changes caused by urea are likely to be a breaking or distortion of the bond between the iron and the protein-donated nitrogenous ligand and are similar to alterations seen with NO complexes of hemoglobin under a variety of conditions.

Highlights

(nitrite reductase) purifiedfrom Pseudomonas aerugi- NO is shared with the iron and to varying degrees with other nom, of its heme &depleted form, and of heme dl in iron-bound ligands
Sephadex G-25 wasobtained from Pharresolution of the high field (g,) resonance whicbhe camnacia Fine Chemicals
Pseudomonas cytochrome oxidase was purified from cultures of P. aeruginosa by the procedure of Gudat et al [4].Heme and after interaction with urseuaggests structural sim- dl was extracted from the enzyme with acidified acetone (0.24 N HC1)

Summary

EXPERIMENTAL PROCEDURES

Puntled Pseudomonas cytochrome oxidase consists of two equivalent subunits, each having a molecular weight of about 6 0 , O O O daltons [4, 5] and each containing one heme dl and one heme c moiety [5] Both types of heme have been oxidase depleted of heme dl,was first dissolved in 0.04 M potassium phosphate, pH 7.0, and dialyzed for 24 h against 2000 volumes of the same buffer before use. The cytochrome c was unfolded by dialysis for 24 h against 2000 volumes of 6 M urea buffered with 0.25 M potassium phosphate, pH 7.0 These solutions contained at most a implicated in the process of electron transfer.

Pseudomonas Cytochrome Oxidase

RESULTS

DISCUSSION