REAL-TIME STOPPED-FLOW SPECTROSCOPY OF HIGH-VALENT MANGANESE-OXO INTERMEDIATES IN MANGANESE SUPEROXIDE DISMUTASE (Mn-SOD) CATALYSIS.

Manganese superoxide dismutase (Mn-SOD) is one of the critical metalloenzymes reciprocally involved in protection from oxidative stress discharge through the dismutation of superoxide radicals (O2-) to oxygen and hydrogen peroxide. The catalytic mechanism was dissected with stopped flow spectroscopy, UV-Vis., EPR, X-ray Ab sorption Spectroscopy (XAS), and DFT calculations using this study. Rapid reduction of Mn(III)-SOD to Mn(IV)=O and Mn(V)=O species was stopped flow kinetics shown (k1 = 1.2 × 108 M-1 s-1) with subsequent slow kinetics (k3 = 3.5 × 106 M-1 s-1), indicative of a partially rate limiting step. XANES edge shifts of +1.5 eV and +3.0 eV, as well as λmax at 420 nm and 440 nm for Mn(IV)=O and Mn(V)=O, respectively, indicated by UV-Vis spectroscopy, provided support for this. The deuterium isotope, pH, and EPR spectra all indicate that proton coupled electron transfer (PCET) occurs with kinetic isotope effect (KIE) = 2. Intermediate stability, as also demonstrated through DFT modelling, and measured redox potentials agreed with the experiment. This work confirms that metalloenzyme catalysis proceeds via a dual pathway mechanism consistent with intermediates that carry high valences. [ABSTRACT FROM AUTHOR]