Rapid-freeze quench technique

EPR spectroscopy in combination with stopped-flow absorption and rapid freeze-quench techniques has been employed (i) to probe flie catalytically relevant oxidation state(s) of MIOX and (ii) to investigate the reaction between MIOX, substrate, and O2. While most other oxygen-activating binuclear non-heme iron enzymes are catalytically active in their fully reduced form, MIOX exhibits a raflier different behavior. In single-turnover reactions of the diferrous recombinant Mus... 

When the fully reduced enzyme is reacted with O2, the 4e reduced native intermediate (NI) is formed. This intermediate has been trapped using a rapid freeze-quench technique and spectroscopically characterized using EPR, absorption, CD, VTVH MCD, and XAS [22,105]. It was shown that the NI is a fully oxidized species with the three Cu(II) centers in the trinuclear site mutually bridged by the product of the full 4e O2 reduction with cleavage of the 0-0 bond. 

Both stopped-flow and rapid freeze quench kinetic techniques show that the substrate reduces the flavin to its hydroquinone form at a rate faster than catalytic turnover Reoxidation of the flavin hydroquinone by the oxidized Fe4/S4 center leads to formation of a unique spin-coupled species at a rate which appears to be rate limiting in catalysis. Formation of this requires the substrate since dithionite reduction leads to flavin hydroquinone formation and a rhombic ESR spectrum typical of a reduced iron-sulfur protein . The appearance of such a spin-coupled flavin-iron sulfur species suggests the close proximity of the two redox centers and provides a valuable system for the study of flavin-iron sulfur interactions. The publication of further studies of this interesting system is looked forward to with great anticipation. 

The single-turnover reaction of MMOH ed with O2 has been monitored by time-resolved spectroscopic techniques. Stopped-flow optical spectroscopic and rapid freeze-quench (RFQ)... 

As discussed further in the following sections, there are other variations of rapid mixing/quench methods in which the enzymatic reaction is terminated by freezing the reaction mixture with liquid isopropane. The frozen sample is then analyzed hy electron paramagnetic resonance (EPR), solid-state NMR, or other spectroscopic techniques such as resonance Raman spectroscopy that can accommodate a solid sample. Perhaps the major limitation for implementation of this methodology is the sensitivity of the spectroscopic method and the requirement for large amounts of enzyme. ... 

In an examination of the literature dealing with the study of non-aqueous solutions, it is striking that there is virtually no experimental procedure for the investigation of matter that has not been employed to study solvation or other processes involving solvent effects. Scarcely does a new method appear in the arsenal of the analyst than it is applied to this field of solution chemistry. This tendency is well shown by the example of typical methods, developed for the investigation of solid substances, such as Mossbauer spectroscopy and ESCA. The application of these to the study of solvation processes was made possible by the elaboration of the technique of quenching solutions by rapid freezing. 

In contrast to sublimation in freeze-drying, evaporation of a solvent is straightforward but may lead to less homogeneity for example, if a multicomponent solution is slowly evaporated, the various constituents crystallize nonuniformly. Thus, the goal of evaporative techniques is to break the solution into small droplets to minimize the volume over which segregation can take place as well as to maximize the surface area for evaporation and then evaporate the solvent as rapidly as possible to quench in the homogeneity of the solution. 

Nearly all materials can be prepared in the amorphous state. The principle involves rapid quenching, which prevents the material from crystallizing in an ordered manner. This can be achieved in a number of ways, including supercooling (cooling below the freezing point), vapour deposition and gelation. The majority of these methods lead to materials which cannot be easily characterized by normal techniques such as X-ray diffraction. 

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