Freeze-quenching

The mechanism of the first half-reaction has been studied by a combination of reductive titrations with CO and sodium dithionite and pre-steady-state kinetic studies by rapid freeze quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics 159). These combined studies have led to the following mechanism. The resting enzyme is assumed to have a metal-bound hydroxide nucleophile. Evidence for this species is based on the similarities between the pH dependence of the EPR spectrum of Cluster C and the for the for CO, deter-... 

However, useful as it is, ligand field theory is not a predictive first principles theory. Thus, it cannot be used to predict a priori the Mossbauer parameters of a given compound. Yet, the need to do so arises fi equently in Mossbauer spectroscopy. For example, if a reaction intermediate or some other unstable chemical species has been characterized by freeze quench Mossbauer spectroscopy and its SH parameters become available, then the question arises as to the structure of the unstable species. Mossbauer spectroscopy in itself does not provide enough information to answer this question in a deductive way. However, the more modest question which structures are compatible with the observed Mossbauer parameters can be answered if one is able to reliably predict Mossbauer parameters... 

The time-dependent, rapid freeze-quench Mossbauer experiments with M. capsulatus (Bath) (51) indicate that decay of the peroxo species proceeds with the concomitant formation of another intermediate, named compound Q. This intermediate, observed in both the M. tri-chosporium OB3b (69, 70) and M. capsulatus (Bath) (51, 71) MMO systems by Mossbauer and optical spectroscopy, decays faster in the presence of substrates. Such behavior indicates that this intermediate is probably on the kinetic reaction pathway for hydroxylation (51, 70). 

The initial oxidation of the d heme by oxygen is followed by chemical events, the interpretation of which was assisted by parallel freeze-quenched EPR measurements. There was one unexpected process detected by optical spectroscopy. This was rereduction of the c heme center by ascorbate on the seconds time scale. This was surprising because the initial reduction by ascorbate took more than 2 hours. 

Francium, binary carbide not reported, 11 210 Franck-Condon effect, 16 69 energy, 21 180, 188, 189 envelopes, 16 80, 89, 90 hot bands, 16 90 factors, 32 47 principle, 21 179, 181 vibronic replica, 35 370 frd redon, 38 412, 414 Freeze quench EPR spectroscopy (FQ-EPR), CODH/ACS, 47 318 Fremy s salt, 33 106 Friedel-Crafts reaction, 17 194 cyclophosphazene, 21 65, 66 Frontier molecular orbitals, heteronuclear gold cluster compounds, 39 378-381 Frozen solutions, MOssbauer spectra in studies of, 15 101-103... 

Several studies have dealt with the influence of lipids on conformational equilibria in cytochrome c via hydrophobic and electrostatic interactions. The binding of sodium dodecyl sulfate monomers and micelles was reported to cause a transition of cytochrome c to a state B2 which is of potential physiological relevance. The interplay between heme only state changes and secondary structure changes was analyzed by freeze-quench and stopped-flow experiments.276 The response of the heme spin state to lipid acyl chains in cytochrome c was... 

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. 

Kim SH, Perera R, Hager LP, Dawson JH, Hoffman BM (2006) Rapid Freeze-Quench ENDOR Study of Chloroperoxidase Compound I The Site of the Radical. J Am Chem Soc 128 5598... 

Spectroscopies are also used to experimentally probe transient species along a reaction coordinate, where often the sample has been rapidly freeze quenched to trap intermediates. An important theme in bioinorganic chemistry is that active sites often exhibit unique spectroscopic features, compared to small model complexes with the same metal ion.8 These unusual spectroscopic features reflect novel geometric and electronic structures available to the metal ion in the protein environment. These unique spectral features are low-energy intense absorption bands and unusual spin Hamiltonian parameters. We have shown that these reflect highly covalent sites (i.e., where the metal d-orbitals have significant ligand character) that can activate the metal site for reactivity.9... 

A Si micromixer was constmcted with seven vertical pillars (10 pm dia.) arranged perpendicular to the flow in a staggered fashion within a 450-pL mixing chamber, as shown in Figure 3.42. Turbulent mixing of sodium azide and horse heart myoglobin was achieved in 20 ps. This provided the fast mixing essential for a downstream freeze-quench procedure to trap metastable intermediates [472]. 

Krebs C, Bollinger JM Jr. Freeze-quench (57,Fe-M6ssbauer spectroscopy trapping reactive intermediates. Photosynth Res. 2009 102 295-304. 

Mitic N, Saleh L, Schenk G, Bollinger JM, Solomon El. Rapid-freeze-quench magnetic circular dichroism of intermediate X in ribonucleotide reductase new structural insight. J Am Chem Soc. 2003 125 11200-1. 

Freeze-quenching technique in combination with ESR and Mossbauer spectroscopy was used for monitoring intermediates in the reaction of substrate free 57Fe-P450C8Itl with peroxy acetic acid (Schunemann et al., 2000). In such a condition, the oxidant oxidized the enzyme active site iron (III) to iron (VI) and Tyr 96 into tyrosine radical, 90% and 10% from the starting material, respectively. Thus the tyrosine residue may be involved in the catalytic process. 

Identification of radical 3 as a species that is present in the steady-state phase of the reaction does not prove that it is an intermediate—it could be a species that is peripheral to the real reaction mechanism. Proof that a species is an intermediate requires a demonstration that it is kinetically competent to participate in the mechanism. In the case of a metastable radical, the usual procedure is to conduct transient kinetic studies using a rapid mixing apparatus equipped to quench samples by spraying them into liquid isopentane. The frozen aqueous samples (snows) from the timed cold quenches are then packed into EPR tubes and analyzed spectroscopically. Simple mixing of enzyme with SAM and lysine followed by freeze-quenching on the millisecond time scale does not work because the activation by SAM takes about 5 s. However, a preliminary mix of enzyme with SAM and [2- C]lysine, aging of the solution for 5 s within the apparatus. 

---