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.
Cytochrome c Oxidase: Structure
The cytochrome oxidases are a wide and heterogeneous family of electron transfer hemoproteins which play a central role in the energy metabolism of aerobic organisms. They catalyze the terminal electron transfer steps in the complex branched respiratory systems of prokaryotes as well as in the mitochondrial respiratory chain [1]. The structural and functional variety of these enzymes is perhaps exemplified by P. denitrificans where, depending on the growth conditions, up to five different terminal oxidases termed aa 3 , cytochrome o, cytochrome a 1 , cytochrome d and cytochrome cd 1 can be synthesized [2]. The differences among the various oxidases are consistent. Copper and heme a, the redox centers present in the mitochondrial enzyme, may be replaced in the bacterial terminal oxidases by different prosthetic groups, while cytochrome c and oxygen may not always be the electron donor and acceptor. Also the integration in the membrane does not appear as a general feature. Depending on their spectroscopic properties and prosthetic groups, the cytochrome oxidases have been arranged in different classes [2, 3]. Nevertheless, the structure and the kinetic competence of most of them remain poorly understood. In this situation, changes of class and nomenclature are not surprising [4, 5]. Only recently the structural organization of these proteins in bacteria began to be elucidated at molecular level. In E. coli the two terminal oxidases, termed cytochrome d and cytochrome o, have been characterized.
Cytochrome oxidase and its derivatives. VI. The CO combining capacity of cytochrome C oxidase.
Summary Ferrous cytochrome a 3 is known to bind CO at pH 7.4, whereas cytochrome a does not do so. It is found that about one-half of the total haem a of cytochrome c oxidase (EC 1.9.3.1) is able to bind CO. This shows that the ratio of cytochrome a to cytochrome a 3 is about 1. The validity of the method was established by showing that haemoglobin bound 1 mole of CO per mole of protohaem. When cytochrome oxidase is kept at pH 10 at room temperature both cytochromes a and a 3 are modified and both the modified cytochromes now react with CO. It is found that 1 mole of haem a of the modified oxidase binds approx. 1 mole of CO. This lends additional support to the result of the experiments with native cytochrome oxidase.