Demonstration of peroxodiferric intermediate in M-ferritin ferroxidase reaction using rapid freeze-quench Mössbauer, resonance Raman, and XAS spectroscopies

Abstract

This chapter presents the advantages and disadvantages of the four methods employed in study. Stopped-flow absorption and RFQ-Mossbauer are suitable to obtain time resolution of species participating in the reaction under investigation. The obvious limitations of RFQ-Mossbauer spectroscopy are that (1) only 57 Fe-enriched proteins can be studied, and (2) very concentrated solutions are required. If these two criteria are met (as in this study) RFQ-Mossbauer spectroscopy is extremely useful because it provides an accurate quantification and detailed characterization of all Fe species involved in the reaction. The chapter describes RFQ-XAS and RFQ resonance Raman, which may be used to obtain detailed information about the geometric and electronic structure of reaction intermediates. For RFQ-resonance Raman spectroscopy, a major problem lies in the fact that the cryosolvent may be a good Raman scatterer, thereby possibly overshadowing true signals from the sample. Liquefied ethane is a suitable cryosolvent because it can be removed from the sample at -80°. As this temperature is significantly higher than the temperature of the quenching bath, it is important to make sure that the reaction intermediate is stable under these conditions.