Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives

G T Babcock,L E Vickery,G Palmer

doi:10.1016/s0021-9258(19)57020-x

Abstract

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).

Highlights

From the Department of Biochemistry, Rice Uniuersity, Houston, Texas 77005, and the SLaboratory of Chemical Biodynamics, University of California, Berkeley, California 94720
The Soret Magnetic circular dichroism (MCD) of the reduced protein is interpreted as the sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a,*+ and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a’+
MCD curve associated with the 600 nm a-band which is reversed to that routinely observed with other hemeproteins in that the arm of negative intensity is to shorter wavelengths

Summary

Introduction

MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. The ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a3 3+. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome as’+; the band associated with cytochrome u2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. It is firmly established that the active center of cytochrome oxidase contains 2 mol of heme a and two atoms of copper (l-3) there is considerable uncertainty regarding the functional relationships between these four metal components [4, 5]

Methods

Results

Conclusion