JBC, Vol. 251, Issue 24, 7907-7919, Dec, 1976
Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives
G. T. Babcock, L. E. Vickery and G. Palmer
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).
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