Freeze-quench resonance raman and electron paramagnetic resonance spectroscopy for studying enzyme kinetics: Application to azide binding to myoglobin

In the present work we have developed a reliable approach for probing the r eaction dynamics of metalloproteins on the millisecond time scale. It is ba sed on the combination of the freeze-quench method with resonance Raman (RR ) and electron paramagnetic resonance (EPR) spectroscopy. The reactions are initiated in a mixing chamber and rapidly quenched at low temperature in l iquid isopentane after variable delay times. The experimental device is des igned in such a way that the same frozen samples can be subsequently studie d by two analytical techniques, thereby providing complementary information about the active site structures of intermediate states of the enzyme. In particular, the present setup permits the measurement of high-quality RR sp ectra despite the interference by the Raman bands of isopentane. With the u se of the azide binding reaction to myoglobin as a test case, it is found t hat RR spectroscopy allows a reliable determination of rate constants, wher eas the quantitative analysis of the EPR spectra is associated with a relat ively high and unavoidable uncertainty mainly due to irreproducible packing of the freeze-quenched samples.