Improved stopped‐flow time‐resolved resonance Raman spectroscopy device for studying enzymatic reactions

Abstract

An improved stopped-flow resonance Raman spectroscopy device was constructed using a stopped-flow mixer with a dead time of 3 ms and a mixing volume of 0.1 mL. The device was tested using myoglobin, where the formation reaction of a high-valent heme species, ferryl-oxo heme, was monitored by time-resolved resonance Raman spectroscopy after mixing a ferric myoglobin solution with a hydrogen peroxide solution. The ferryl-oxo heme formation rate constant obtained by Raman spectroscopy is in good agreement with the rate constant obtained by conventional stopped-flow absorption spectroscopy for the same reaction under the same conditions. It is proved by these results that the present device is generally applicable to enzyme-substrate reactions with a significantly higher time resolution than previously reported.

Reisomerization of retinal represents a molecular switch mediating Na+ uptake and release by a bacterial sodium-pumping rhodopsin

“…This cryo-Raman method allowed us to accumulate the specific photointermediates by controlling the temperature and observe their structural transitions based on the time-dependent Raman spectra. Because of the sufficient accumulation of the photointermediates at low temperatures, we can apply the near-IR Raman excitation to measure the spectra without the fluorescence interference or the unfavorable side products, which often complicate the interpretation of the time-resolved resonance Raman spectra ( 15 , 16 ). We successfully observed the vibrational spectra of O1, O2, and N. The spectral features indicated that O1 and O2 have the 13- cis and all- trans forms of the RSB chromophore, respectively.…”

Recent advances in linear and nonlinear Raman spectroscopy. Part XII

“…The device was tested using myoglobin, where the formation reaction of a high‐valent heme species and ferryl‐oxo heme, was monitored by time‐resolved resonance Raman spectroscopy after mixing a ferric myoglobin solution with a hydrogen peroxide solution. These results show that the new device is generally applicable to enzyme‐substrate reactions with a significantly higher time resolution than has previously been reported …”