Formation of high spin cytochrome a in isolated cytochrome c oxidase.

The effect of temperature on the electronic structure of cytochromes a and a3 has been determined at two different concentrations of the solubilizing detergent, N-dodecyl beta-D-maltoside. In each case, an incubation temperature of 40 degrees C resulted in a 20-30% increase in the 430-nm conformer of cytochrome a3 when compared with a 20 degrees C control, but the higher detergent concentration led to additional formation of a new charge transfer band at 630 nm. Induction of this new feature was correlated with a loss of alpha-band intensity, a blue shift in the Soret absorbance, and a decrease in the Soret magnetic circular dichroism. The absorbance changes were associated with isosbestic wavelengths at 613, 581, 563, 453, and 416 nm, indicating a two-state electronic conversion. The loss of magnetic circular dichroism intensity and the lack of an isosbestic point between 630 and 655 nm were consistent with a selective effect on the electronic structure of ferricytochrome a. Comparison of these spectral changes with the model heme a studies of Carter and Palmer (Carter, K., and Palmer, G. (1982) J. Biol. Chem. 257, 13507-13514) provides evidence for a low to high spin transition in cytochrome a in which one of the axial Fe-N bonds is either broken or sterically strained. This selective effect on cytochrome a and the associated thermal resistance of the a3-CuB domain are discussed in terms of the 4-helix bundle motif that has been recently proposed for the catalytic site of cytochrome c oxidase.