Application of resonance Raman spectroscopy for interrogation of cryoradiolytically reduced oxygenated heme proteins

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Resonance Raman (rR) spectroscopy is generally useful to interrogate unstable intermediates that arise in the enzymatic cycles of heme enzymes, such as peroxo-, hydroperoxo- and ferryl species. While it is typically difficult to trap these elusive intermediates in solution, the cryoradiolysis approach offers a way to successfully overcome these obstacles. The present work employs modified derivatives of horse heart myoglobin to demonstrate the methodology and utility of a combination of rR and cryoradiolysis for detection and structural characterization of Fe―O―O, Fe―O―O―H and FeO fragments of such reactive species. First, the effect of active site mutations on the Fe―O―O fragments was investigated; specifically, the His64Leu, Val68Ser and Ile107Ala in the distal pocket and Ser92Ala, Ser92Leu and His97Phe on the proximal side. The current work has permitted documentation of the wavenumbers of the ν(Fe―O) stretching modes of the relatively unstable dioxygen adducts of these mutants, some of them previously unreported. Significantly, applying the cryoradiolysis approach has yielded previously unavailable vibrational spectroscopic data for the peroxo-, hydroperoxo and ferryl derivatives of these mutants. Of the six mutations investigated, only two, the H64L and V68S, replacements had a significant effect on the structure and stability of these unstable intermediates. In addition to these manipulations of the heme active site environment, the study of myoglobins bearing modified hemes was also undertaken; namely, myoglobins reconstituted with mesoheme and diformyl deuteroheme. The results show that the heme equatorial electronic effects significantly influence the stability and internal modes of the Fe―O―O and FeO fragments of these unstable oxy intermediates. Copyright © 2016 John Wiley & Sons, Ltd.