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.
The low-temperature spectral properties of mammalian cytochromes oxidase. II. The enzyme isolated from beef-heart mitochondria.
Abstract A series of low-temperature spectra of isolated cytochrome oxidase are presented. 1. 1. A technique for the selective reduction of cytochrome a with dithionite is given. 2. 2. The reduced minus oxidized difference spectrum of cytochrome a shows a 601 mμ α-peak and a split Soret band with maxima at 447 and 442 mμ for ferrous a and a Soret absorption band at 426 mμ for ferric a. 3. 3. The cytochrome a3 difference spectrum is quite different. Its ferrous α-peak is found at 604 mμ and its Soret band has a single maximum at 444 mμ. The ferric γ-band is at 412 mμ. 4. 4. Sulfide, like cyanide, was found to react with ferricytochrome a3. 5. 5. Azide was found to react with ferrocytochrome a. The 601 mμ peak of ferrous a was shifted to 597 mμ and the split Soret band to 447 and 440 mμ. 6. 6. Under anaerobic conditions the azide effect was reversed, indicating that ferrocytochrome a can exist in two forms differing in reactivity toward azide. The form which reacts with azide is labile and in the absence of active turnover is converted to the form which does not react with azide. 7. 7. ‘Oxygenated’ cytochrome oxidase showed no Soret band splitting at low temperature.
Diagnosis of the σ-, π- and (σ+π)-Aromaticity by the Shape of the NICSzz-Scan Curves and Symmetry-Based Selection Rules
The NICSzz-scan curves of aromatic organic, inorganic and “all-metal” molecules in conjunction with symmetry-based selection rules provide efficient diagnostic tools of the σ-, π- and/or double (σ + π)-aromaticity. The NICSzz-scan curves of σ-aromatic molecules are symmetric around the z-axis, having half-band widths approximately less than 3 A with the induced diatropic ring current arising from Tx,y-allowed transitions involving exclusively σ-type molecular orbitals. Broad NICSzz-scan curves (half-band width approximately higher than 3 A) characterize double (σ + π)-aromaticity, the chief contribution to the induced diatropic ring current arising from Tx,y-allowed transitions involving both σ- and π-type molecular orbitals. NICSzz-scan curves exhibiting two maxima at a certain distance above and below the molecular plane are typical for (σ + π)-aromatics where the π-diatropic ring current overwhelms the σ-type one. In the absence of any contribution from the σ-diatropic ring current, the NICSzz(0) value is close to zero and the molecule exhibits pure π-aromaticity.