Formation of a tyrosyl radical intermediate in Proteus mirabilis catalase by directed mutagenesis and consequences for nucleotide reactivity

Proteus mirabilis catalase (PMC) belongs to the family of NADPH binding cat alases. The function of NADPH in these enzymes. is still a matter of debate . This study presents the effects of two independent phenylalanine mutation s (F194 and F215), located between NADPH and heme in the PMC structure. The phenylalanines were replaced with tyrosines which we predicted could carry radicals in a NADPH-heme electron transfer. The X-ray crystal structures o f the two mutants indicated that neither the binding site of NADPH nor the immediate environment of the residues was affected by the mutations. Measur ements using H2O2 as a substrate confirmed that the variants were as active as the native enzyme. With equivalent amounts of peroxoacetic acid, wild-t ype PMC, F215Y PMC, and beef liver catalase (BLC) formed a stable compound I, while the F194Y PMC variant produced a compound I which was rapidly tran sformed into compound II and a tyrosyl radical. EPR studies showed that thi s radical, generated by the oxidation of Y194, was not related to the previ ously observed radical in BLC, located on Y369. In the presence of excess N ADPH, compound I was reduced to a resting enzyme (k(obs) = 1.7 min(-1)) in a two-electron process: This was independent of the enzyme's origin and did not require any thus far identified tyrosyl radicals. Conversely, the pres ence of a tyrosyl radical in F194Y PMC greatly enhanced the oxidation of re duced beta -nicotinamide mononucleotide under a steady-state H2O2 flow with observable compound II. This process could involve a one-electron reductio n of compound I via Y194.