Chicken liver sulfite oxidase. Kinetics of reduction by laser-photoreduced flavins and intramolecular electron transfer

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Abstract
Laser flash photolysis was used to study the reaction of photoproduced 5-deazariboflavin (dRFH .cntdot.), lumiflavin (LFH .cntdot.), and riboflavavin (RFH .cntdot.) semiquinone radicals with the redox centers of purified chicken liver sulfite oxidase. Kinetic studies of the native enzyme with dRFH .cntdot. yielded a second-order rate constant of 4.0 .times. 108 M-1 s-1 for direct reduction of the heme and a first-order rate constant of 310 s-1 for intramolecular electron transfer from the Mo center to the heme. The reaction with LFH .cntdot. gave a second-order rate constant of 2.9 .times. 107 M-1 s-1 for heme reduction. Reoxidation of the reduced of the reduced heme due to intramolecular electron transfer to the Mo center gave a first-order rate constant of 155 s-1. The direction of intramolecular electron transfer using dRFH .cntdot. and LFH .cntdot. was independent of the buffer used for the experiment. The different first-order rate constants observed for intramolecular electron transfer using dRFH .cntdot. and LFH .cntdot. are proposed to result from chemical differences at the Mo site. Flash photolysis studies with cyanide-inactivated sulfite oxidase using dRFH .cntdot. and LFH .cntdot. resulted in second-order reduction of the heme center with rate constants identical with those obtainedwith the native enzyme, whereas the first-order intramolecular electron-transfer processes seen with the native enzyme were absent. The isolated heme peptide of sulfite oxidase gave only second-order kinetics upon laser photolysis and confirmed that the first-order processes observed with the native enzyme involve in the Mo site. The flash-induced difference spectrum of native sulfite oxidase using dRFH .cntdot. and LFH .cntdot. resulted in absorbance increases in the 530-570-nm region of the spectrum that were not present in the static difference spectrum of the enzyme. These absorbances and proposed to be associated with the Mo center.