Cytochrome oxidase and its derivatives. IX. Spectrophotometric studies on the rapid reaction of ferrous cytochrome c oxidase with molecular oxygen under conditions of complete and partial oxygenation

Abstract

1. 1. The reaction of ferrocytochrome oxidase with molecular oxygen (15 μM) has a half-time of about 3 msec. 2. 2. 100 msec after 2 μM ferrous oxidase is mixed with 15 μM oxygen the difference spectrum of the ferrous enzyme minus the mixture shows a minimum in the Soret region at 420 mμ. The difference spectrum of the “oxygenated” compound has a minimum at 421–425 mμ while that of the ferric oxidase lies at 411–412 mμ. Thus under these conditions a mixture of “oxygenated” and ferric forms is produced with the “oxygenated” form predominating. 3. 3. By observing the absorption changes at the wavelength pair 428-418 mμ (in a stop-flow dual wavelength spectrophotometer) it is possible to distinguish between “oxygenated” and ferric oxidase formation in the early stages of the reaction between ferrous oxidase and oxygen. When insufficient oxygen is added so that only 10–30% of the ferrous enzyme reacts with oxygen the product is predominantly ferric oxidase with, on the average, about 30% “oxygenated” compound. The Soret band of this mixture has a broad maximum extending from 426 to 418 mμ. Similar results were obtained with cytochrome c-deficient electron transport particle. 4. 4. Vigorous aeration of ferrous oxidase produces “oxygenated” compound only, whereas, mixing with insufficient oxygen produces a mixture of ferric and “oxygenated” forms. It is suggested that “oxygenated” oxidase is formed by the interaction of the primary a 3 FeO 2 complex with ferrocytochrome a, whereas ferric cytochrome oxidase ( a 3‴ + a‴) results when the primary complex can interact with ferrocytochrome a 3 as well as with ferrocytochrome a. 5. 5. On reduction of the ferric enzyme with dithionite the absorption changes at 428-418 mμ show an initial decreased absorbance followed by the expected increase. The initial absorbance decrease is due to the fast reduction of cytochrome a which has a Soret band at 426 mμ in the ferric form. This is followed by the slower reduction of cytochrome a 3 with its Soret band at 414 mμ causing an absorbance increase at 428-418 mμ.