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

doi:10.1016/0005-2795(76)90118-5

Biochimica et Biophysica Acta (BBA) - Protein Structure

Volume 446, Issue 1, 28 September 1976, Pages 277-286

https://doi.org/10.1016/0005-2795(76)90118-5 Get rights and content

Abstract

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_{e_aq⁻ + methemoglobin}  6.5 ± 1 · 10¹⁰M⁻¹·s⁻¹·k_{e_aq⁻ + 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.

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Reaction of horseradish peroxidase A₂ and C with superoxide anion (O₂⁻) has been studied using pulse radiolysis technique. Peroxidase C formed Compound I and an oxy form of the enzyme due to reaction of ferric enzyme with hydrogen peroxide (H₂O₂) and O₂⁻, respectively. At low concentrations of O₂⁻ (<1 μM), O₂⁻ reacted with ferric peroxidase C nearly quantitatively and formation of H₂O₂ was negligible. The rate constant for the reaction was found to be increased below pH 6 and this phenomenon can be explained by assuming that HO₂ reacts with peroxidase C more rapidly than O₂⁻. In contrast, the formation of oxyperoxidase could not be detected in the case of peroxidase A₂ after the pulse, and only Compound I of the enzyme was formed. Peroxidase A₂, however, produced the oxy form upon aerobic addition of NADH, suggesting that O₂⁻ can also react with peroxidase A₂ to form the oxy form. The results at present indicate that the rate constant for the reaction of O₂⁻ with peroxidase A₂ is smaller than 10³ M⁻¹·s⁻¹.
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^☆: Part of this work was performed at the Hahn Meitner Institute in Berlin.

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

Abstract

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