Microsecond freeze-hyperquenching: development of a new ultrafast micro-mixing and sampling technology and application to enzyme catalysis

A novel freeze-quench instrument with a characteristic much less thandead-timemuch greater than of 137 +/- 18 lis is reported. The prototype has several key features that distinguish it from conventional freeze-quench devices and provide a significant improvement in time resolution: (a) high operating pressures (up to 400 bar) result in a sample flow with high linear rates (up to 200 in s(-1)); (b) tangential micro-mixer with an operating volume of similar to 1 nl yields short mixing times (up to 20 mus); (c) fast transport between the mixer and the cryomedium results in short reaction times: the ageing solution exits the mixer as a free-flowing jet, and the chemical reaction occurs "in-flight" on the way to the cryomedium; (d) a small jet diameter ( similar to 20 mum) and a high jet velocity ( similar to 200 m s(-1)) provide high sample-cooling rates, resulting in a short cryofixation time (up to 30 lis). The dynamic range of the freeze-quench device is between 130 lis and 15 ins. The novel tangential micro-mixer efficiently mixes viscous aqueous solutions, showing more than 95% mixing at eta less than or equal to 4 (equivalent to protein concentrations up to 250 mg ml(-1)), which makes it an excellent tool for the preparation of pre-steady state samples of concentrated protein solutions for spectroscopic structure analysis. The novel freeze-quench device is characterized using the reaction of binding of azide to metmyoglobin from horse heart. Reaction samples are analyzed using 77 K optical absorbance spectroscopy, and X-band EPR spectroscopy. A simple procedure of spectral analysis is reported that allows (a) to perform a quantitative analysis of the reaction kinetics and (b) to identify and characterize novel reaction intermediates. The reduction of dioxygen by the bo(3)-type quinol oxidase from Escherichia coli is assayed using the MHQ technique. In these pilot experiments, low-temperature optical absorbance measurements show the rapid oxidation of heme o(3) in the first 137 mus of the reaction, accompanied by the formation of an oxo-ferryl species. X-band EPR spectroscopy shows that a short-living radical intermediate is formed during the oxidation of heme o(3). The radical decays within similar to 1 ins concomitant with the oxidation of heme b, and can be attributed to the P-M reaction intermediate converting to the oxoferryl intermediate F. The general field of application of the freeze-quench methodology is discussed. (C) 2004 Elsevier B.V. All rights reserved.