Cytochrome Oxidases of Pseudomonas Aeruginosa 



317 



to 'turnover' of the oxidase is negligible at the time of measurement. Furthermore 

 the time of maximal oxidation of cytochromes a and a^ overlaps sufficiently, so that 

 the time of measurement of the maxima is not critical. When cytochrome c is present, 

 its concentration is measured at the time of the maximum of cytochromes a and a-^ 

 since cytochrome c is not simultaneously maximal. /7-Phenylenediamine plus ascor- 

 bate, ascorbate alone, or cytochrome c plus ascorbate was used as the reductant. 



The experimental results are of the kind indicated by Fig. 1 which shows the 

 variation of the spectrophotometric effect with the oxidase concentration. It is 

 apparent that the physical effect is maximal at a cytochrome a^ concentration very 

 nearly equal to that of the oxidizing equivalents added. In Fig. 2, the variation of the 



Z 

 UJ 



e I 



d _*' 



CD T 



liJ 

 O 



o 



»- 



UJ 



z 

 o 



< 

 2 



o 



X 



10 20 30 40 50 60 

 CONCENTRATION OF O2 (oxidant equivalent) 



Fig. 2. An example of a magnetometric titration of cytochromes a and O3 

 with oxygen. Reaction at 100 msec after mixing at 29°C. Concentrations 

 as follows: cytochrome 03, 30 /(m; cytochrome c, 6 /<m; ascorbate, 100 mM. 



magnetometric effect with oxygen concentration again shows an approximate equiva- 

 lence of cytochrome O3 and the oxidizing equivalents added. Table 1 summarizes the 

 spectroscopic results. 



In all experiments, the cytochrome concentration was calculated on the basis that 

 the change of extinction coefficient between the reduced and the oxidized forms, as 

 measured at 605 m/t and 630 m//, is 16 cm^^ x mM~^ (Chance, /. biol. Chem. 202, 

 407, 1953). 



Table 1. Summary of results in oxygen-cytochrome oxidase 

 stoichiometry 



25°C, 10 mM /7-phenylenediamine, pH 7-2, 40 mM ascorbate 



