Rapid 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-... 

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). 

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... 

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. 

CEMS = conversion electron Mossbauer spectroscopy DFT = density functional theory EFG = electric field gradient EPR = electron paramagnetic resonance ESEEM = electron spin echo envelope modulation spectroscopy GTO = Gaussian-type orbitals hTH = human tyrosine hydroxylase MIMOS = miniaturized mossbauer spectrometer NFS = nuclear forward scattering NMR = nuclear magnetic resonance RFQ = rapid freeze quench SAM = S -adenosyl-L-methionine SCC = self-consistent charge STOs = slater-type orbitals TMP = tetramesitylporphyrin XAS = X-ray absorption spectroscopy. 

Microsecond Freeze-HyperQuench. RFQ, Rapid Freeze-Quench. 

Calculations based on p = lOOOkgm" and r = lO kgm RFQ rapid freeze-quench. 

Ascorbate Much faster, reaction within the (18) Rapid freeze- -quench EPR experiments indicate that type-2 copper is (18)... 

The anaerobic reduction of the trinuclear copper center for ascorbate oxidase with different substrates presents a distinct picture. The reaction with reductate is monophasic with a unimolecular rate constant of 100 sec (18), independent of pH. Rapid freeze-quench EPR experiments indicate that the type-2 EPR signal vanishes more slowly 18). The pulse radiolysis studies of the radicals of lumiflavin, deazaflavin, CO2 ", and MV at pH 7.0 129,130) showed a biphasic behavior with an initial, faster reaction k = 97-127 sec " ) and a final, slower reaction k 2 sec" ) 129). Different results have been obtained by Farver and Pecht 130) with CO2 " as a substrate. They found a triphasic reaction with unimolecular rate constants k = 201 sec S 2 = 20 sec", and ks = 2.3 sec. The first constant is twice that in a study by Kyritsis et al. 129), whereas the third constant is identical. The second constant was not observed in the study. 

The finding from rapid-freeze-quench EPR experiments, that the reduction of the type 2 copper is slow compared with that of the type 1 copper, is analogous to the behavior noted for tree laccase at higher pH values (50). In this enzyme the slow reduction of the type 2 center is linked to the inhibition of the type 3 reduction. In ascorbate oxidase, however, reduction of the type 3 copper pairs proceeds despite the slow reduction of the type 2 copper, suggesting that the two electrons necessary for the proposed intramolecular reduction of the two type 3 copper pairs can be transferred from two of the three type 1 copper centers, without involving the type 2 center in any redox process. 

Transient kinetics by spectrophotometry and rapid freeze-quench EPR showed the appearance of cob(II)alamin and the radical cation at the same rate, about 3 s . Further studies of deuterium partitioning between [1- H2]HEH and 5 -deoxyadenosine were consistent with direct and reversible deuterium transfer between HEH and the 5 -deoxyadenosyl radical. Reversibility was proven by the appearance of multiply deuterated 5 -deoxyadenosine at all stages of the reaction. 

A series of rapid chemical quench experiments under single enzyme turnover conditions using radiolabeled S3P or PEP revealed that the tetrahedral ketal phosphate enzyme intermediate was formed as a new peak upon HPLC analysis with detection of the radiolabel. The time course revealed that the formation of the tetrahedral intermediate species paralleled the disappearance of PEP substrate and formation of the EPSP product thus establishing that it was a kinetically competent species. Isolation of the tetrahedral ketal phosphate intermediate using C-2 PEP and S3P as substrates coupled with rapid chemical quench was carried out in conjunction with H-, C-, and P- NMR to provide a definitive structure proof Thus with these studies we have satisfied the criteria for a true reaction intermediate in terms of a chemically plausible mechanism, structure proof, and kinetic competence. Additional studies support the mechanism for EPSP synthase described (Scheme 4, pathway a) including observation of the intermediate bound to the enzyme at internal equilibrium using solution NMR and C-2 PEP as well as using rapid freeze-quench/solid-state NMR studies. ... 

Scheme 10-5 shows the simplest version for a possible mechanism of enzyme activation. However, this mechanism does not reveal much about the control of the S-C bond cleavage within the intermediate sulfuranyl radical. Spontaneous cleavage of this radical within the active site is not unlikely if one takes the great instabihty of such radicals into account. Furthermore it has to be emphasized that no direct evidence for intermediate free radicals, such as the sulfuranyl radical or the 5 -deoxyadenosyl radical, is available yet. Whether these radicals are true intermediates or not need to be demonstrated, by using rapid freeze quench EPR methods for example. 

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