Mutations have been introduced at residues Arg-38 or His-42 in horseradish peroxidase isoenzyme C (HRPC) in order to probe the role of these key distal residues in the reaction of ferrous HRPC with dioxygen. The association and dissociation rate constants for dioxygen binding to His-42 --> Leu, His-42 --> Arg, Arg-38 --> Leu, Arg-38 --> Lys, Arg-38 --> Ser, and Arg-38 --> Gly variants have been measured using stopped-flow spectrophotometry. Replacement of His-42 by Leu or Arg increases the oxygen binding rate constant by less than an order of magnitude, whereas changing the polar distal Arg-38 causes increases of more than 2 orders. These results demonstrate that His-42 and Arg-38 impede the binding of dioxygen to ferrous HRPC, presumably by steric and/or electrostatic interactions in the distal heme cavity. Recombinant HRPC oxyperoxidase reverted slowly to the ferric state with no spectrophotometrically detectable intermediates and with an apparent first-order rate constant of 9.0 x 10(-3) s(-1), which is essentially the same as that for the native, glycosylated enzyme. This reaction was accelerated when His-42 was replaced by Leu or Arg (k(decay) = 0.10 and 0.07 s(-1), respectively) presumably due to the loss of the hydrogen bond between the His-42 imidazole and the bound dioxygen. Substitution of Arg-38 by Leu, Lys, or Gly also produced a less stable oxyperoxidase (k(decay) = 0.22, 0.20, and 0.58 s(-1), respectively). However, with the Arg-38 --> Ser variant, a transient intermediate, proposed to be a ferric-superoxide complex, was detected by rapid-scan stopped-flow spectrophotometry during the conversion of oxyperoxidase to the ferric state. This variant also exhibits an unusually high affinity for dioxygen. It is proposed that Arg-38 interacts with the bound dioxygen to promote superoxide character, thereby stabilizing the oxyperoxidase state and making the binding of dioxygen to ferrous HRPC essentially irreversible. We conclude that Arg-38 and His-42 not only promote the heterolytic cleavage of bound hydrogen peroxide to form compound I but also decrease the lability of the ferrous enzyme-dioxygen complex in order to suppress the formation of the inactive ferrous state.