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.
Some peculiarities of the steady-state kinetics of electron transfer in submitochondrial particles. A kinetic model based on the idea of activation on the respiratory chain induced by electron transfer.
Abstract 1. The dependence of the steady-state levels of reduced cytochromes on the rate of electron transfer in submitochondrial particles has been studied. 2. The modified Pring equation (M. Pring, in F. Heinmets and L. D. Cady, Concepts and Models in Biomathematics, Dekker, New York, 1969, p. 75) has been used to calculate the rate constants for the interaction of adjacent cytochromes. The constants have been shown to increase linearily with increasing electron transfer rate in the steady state. 3. In order to explain these results a kinetic model has been suggested, based on the assumption that the respiratory chain exists in activated and non-activated forms the relationship of which depends on the rate of electron transfer. 4. The kinetic model gives an equation for the dependence of the steady-state levels of reduced cytochromes upon the rate of electron transfer. 5. The equation allows one to find the rate constants of interaction of the cytochromes (s−1) in the activated and non-activated respiratory chains. The values of the constants agree satisfactorily with the data of Pring and Chance obtained from non-stationary kinetics.
Azide inhibition of mitochondrial electron transport. I. The aerobic steady state of succinate oxidation.
Abstract The azide inhibition of the succinate oxidase activity of rat-liver mitochondria is specific for active (State 3) respiration with no observable inhibition of resting (State 4) respiration. In the range of azide concentrations which inhibit State 3 to rates less than those of State 4, a negative control of respiration by ADP and inorganic phosphate is observed. The inhibition is specific for a site between cytochromes a and a 3 , causing a crossover between these two cytochromes with cytochrome a becoming reduced and cytochrome a 3 remaining highly oxidized. Trapped steady-state difference spectra at liquid nitrogen temperatures show that the reduced cytochrome a in the azide-inhibited system has an α band at 596 mμ, 6 m μ displaced from its usual position at 602 mμ. The azide inhibition is released by uncouplers of oxidative phosphorylation such that the uncoupled respiration requires up to ten times as much azide as does coupled (State 3) respiration for comparable inhibition. The release of inhibition by uncouplers occurs with no change in the steady-state concentration of reduced cytochrome a 596 and the increased respiration is attributed to an increased rate of oxidation of the cytochrome a 596 . This cytochrome is postulated to be either an intermediate in electron transport and energy conservation reactions or an azide compound of such an intermediate.