One electron reduction of metmyoglobin and methemoglobin and the reaction of the reduced molecule with oxygen

We have used the pulse radiolysis technique to reduce with solvated electrons (e_aq^-) a single Fe(III) site in methemoglobin and metmyoglobin. The reduction process was followed spectrophotometrically and the reactions rate constants were measured: k_{eaq^- + methemoglobin} 6.5 ± 1 · 10¹⁰M^-1·s^-1·k_{ea q^- + metmyoglobin} 2.5 ± 0.3·10¹⁰M^-1·s^-1. Approx. 60% of the e_aq have reacted with the hemin group, and the rest of the e_aq^- have probably reacted with the globin moiety. We followed the reaction of the reduced proteins to yield the oxyderivatives and measured the rate constants of the oxygenation processes k_{reduced methemoglobin + O2} 2.6 ± 0.6·10⁷ M^-1·s^-1 and k_myoglobin + O₂ 1.8 ± 0.2·10⁷ M^-1 ·s^-1, All the rate constants were measured at pH 6.8, I 0.004, T 22 ± 2 °C. The high rate constant for reduced methemoglobin indicates that one-site-reduced methemoglobin is probably in the R state, as predicted for methemoglobin from X-ray analysis. The spectra of the reduced and oxygenated species were measured under similar conditions at λ 450-650 nm. We were able to follow slight changes in the micro-second time scale, these changes were attributed to conformational changes. We were not able to detect any reaction between the radical O₂^- and the hemin group (which would result in a complex such as heme -O₂). This may be due to kinetic reasons.