Biochemical and biophysical studies on cytochrome aa3. VI. Reaction of cyanide with oxidized and reduced enzyme

Abstract

1. 1. The reaction of cyanide with cytochrome aa 3 in the fully oxidized and reduced states has been studied. In both cases a single molecule of cyanide is bound reversibly per molecule of aa 3 (that is, 1 mole cyanide per 2 equivalents of haem a). 2. 2. The difference spectrum of fully formed ferric aa 3-cyanide complex minus oxidizid enzyme with maxima at 432, 540 and 585 nm resembles that of other cyanide ferrichaemoproteins; it appears to be a low-spin complex of cytochrome a 3. 3. 3. The equilibrium constant for complex formation with ferric aa 3 ( K D ) was approximately 1 μM; equilibration at 4° took several days as the rate constant for cyanide binding at low cyanide concentrations is only 1.8 M −1·sec −1 (pH 7·4, 21°). At high cyanide concentrations, the rate of spectroscopic complex formation becomes independent of cyanide with a first order constant of 0.018 sec −1. 4. 4. The difference spectrum obtained on addition of cyanide to ferrous aa 3 shows a peak at 587 nm but only a small diminution of absorbance at 445 nm. The equilibrium constant for complex formation with the ferrous enzyme was 100 μM; the rate constant for cyanide binding was 150 M −1·sec −1, apparently representing a simple bimolecular step. 5. 5. The cytochrome aa 3-azide complex reacts more rapidly with cyanide than does the free ferric enzyme; the rate constant for cyanide displacement of azide is 25 M −1·sec −1. The rate limiting step at high cyanide concentrations has the same velocity in the precence as in the absence of azide. 6. 6. The reaction of ferric enzyme with cyanide is interpreted in terms of a two-step reaction involving an initial weak binding to the protein ( K′ α of 10 mM) followed by binding to the a 3 haem. A conformational change accompanying azide binding is proposed that facilitates the subsequent initial weak binding of cyanide ( K′ α ( in presence of N 3 −) = 0.7 mM).