Optical rotatory dispersion and circular dichroism of cytochrome oxidase.
Optical rotatory dispersion of the oxidized and the reduced cytochrome oxidase has been measured from 196 to 550 nm. The experimental data have been analyzed in order to separate the extrinsic cotton effects from the rotations of the protein backbone. From the resolution, two cotton effects of similar sign are obtained in the Soret region. For the reduced enzyme, the cotton effect centered at 446.5 nm shows a molar amplitude of 192 k-degrees with a band width of 5.3 nm and rotational strength of 0.25 Debye magneton; the second cotton effect centered at 444 nm shows a molar amplitude of 45 k-degrees with a band width of 12.9 nm and rotational strength of 0.14 Debye magneton. Upon oxidation, the centers of these cotton effects shift to 432 and 423.7 nm. The amplitudes become 114 and 122 k-degrees with band widths of 6.1 and 11.2 nm and rotational strengths of 0.18 and 0.35 Debye magneton, respectively. In addition, a less defined third cotton effect centered at 376 nm appears with an amplitude of 28 k-degrees, band width of 18 nm, and rotational strength of – 0.15 Debye magneton. The circular dichroic curves constructed from the optical rotatory dispersion data by the Kronig-Kramers transform are in general agreement with the experimentally determined spectra. 
 
 
 
The backbone rotations are analyzed and expressed in the terms of Shechter-Blout's parameters. The effective helicity thus computed is 50% for the reduced and 44.5% for the oxidized form. 
 
 
 
The sources of the Soret cotton effects are discussed along with the possibilities of the components present (cytochromes a and a3 and copper), the intrinsic property of the Soret transition, possible splitting of the Soret band, interactions with the protein helix, and heme-heme and heme-copper couplings. The fact that the optical rotatory dispersion and circular dichroism behavior is dependent upon the oxidation state of cytochrome oxidase could arise either from an altered conformation of the protein backbone and/or from an altered interaction with the heme group. Regardless of the possibilities, the geometrical conformation of cytochrome oxidase and the electronic relation between the prosthetic group and the apoenzyme is dependent upon the oxidation state of the enzyme.
Conformation changes in cytochrome oxidase as studied by sedimentation difference.
Abstract Sedimentation difference measurements show that a conformational change occurs in cytochrome oxidase upon reduction. The nature of the change in the sedimentation coefficient indicates an expansion of the molecule when the oxidized enzyme is reduced. A much smaller conformational change is detected when the a2a23CO and a2a23 and a3a23CO species are compared, indicating a relationship between conformation and the oxidation state of heme a.
Azide inhibition of mitochondrial electron transport. II. Spectral changes induced by azide.
Abstract Azide inhibition of coupled mitochondrial transport is accompanied by spectral changes which indicate that the cytochrome a3 is oxidized and cytochrome a reduced. The cytochrome a α absorption band is shifted to shorter wavelengths in the azideinhibited system. This shift in the α absorption band can be reversed by conditions leading to reduction of cytochrome a3 such as uncouplers and anaerobiosis, or terminal inhibitors such as sulfide, cyanide or CO. Titrations of the azide-induced spectral changes indicate the binding of one azide molecule in the complex, and that the dissociation constant is experimentally indistinguishable from the uncompetitive inhibitor constants for inhibition of State 3 respiration. The azide inhibition is postulated to involve the formation of a reduced cytochrome a azide compound which is unstable in the presence of reduced cytochrome a3.