The formation of the primary hydrogen peroxide compound (compound I) of Pseudomonas cytochrome c peroxidase as a function of pH.

The effect of pH on the stability and overall catalytic activity of half-reduced Pseudomonas cytochrome c peroxidase was studied over the pH range 3.5-8. The stability of the enzyme as deduced from 40 s incubation experiments is virtually unaffected by pH. However, there is a bell-shaped pH dependence for the overall catalytic reaction using H2O2 as oxidizing substrate and cytochrome c-551 as reducing substrate with maximum turnover rate of pH 6. The effects of pH on (1) rate of reduction of the totally ferric enzyme by reduced azurin over the pH range 3.5-8 and (2) the rate of compound I formation from the half-reduced enzyme and hydrogen peroxide over the pH range 4-8 were also investigated. The reduction reaction rate also appears bell-shaped with optimum rate at pH 5.6. The rate of compound I formation is virtually pH independent above pH 5 but drops dramatically as the pH is lowered from 5 to 4. The influence of an ionization with apparent pKa value of 4.4 is implicated in compound I formation. This enzyme acid group must be deprotonated for compound I formation to occur suggesting the importance of base catalysis.