Electron rapid freeze-quench

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... 

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. 

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. 

ACP = acyl carrier protein ACPA D = ACPA desat-urase AlkB = octane 1-monooxygenase AOX = alternative oxidase DMQ hydroxylase = 5-demethoxyquinone hydroxylase EXAFS = extended X-ray absorption fine structure spectroscopy FMN = flavin mononucleotide FprA = flavoprotein A (flavo-diiron enzyme homologue) Hr = hemerythrin MCD = magnetic circular dichroism MME hydroxylase = Mg-protophorphyrin IX monomethyl ester hydroxylase MMO = methane monooxygenase MMOH = hydroxylase component of MMO NADH = reduced nicotinamide adenine dinucleotide PAPs = purple acid phosphatases PCET = proton-coupled electron transfer, PTOX = plastid terminal oxidase R2 = ribonucleotide reductase R2 subunit Rbr = rubrerythrin RFQ = rapid freeze-quench RNR = ribonucleotide reductase ROO = rubredoxin oxygen oxidoreductase XylM = xylene monooxygenase. 

Stopped-flow UV-vis absorption and rapid freeze-quench (RFQ) EPR and Mossbauer studies have shown that the reaction pathway diagrammed in Figure 4 for formation of the tyrosyl radical is essentially accurate except that the diiron(IV) species labeled Q in Figure 4 has never been detected in R2. Instead, an intermediate labeled U (not shown in Figured), occurring prior to X, has properties consistent with a protonated tryptophan cation radical. This radical may shuttle an electron from an external donor to the diiron site in order to reach intermediate In... 

A completely different set of phenomena are observed when the samples are frozen rapidly. If we quench a sample in liquid water at a rate of, say, 1000 K/sec, it is likely that we will not see the thermodynamic phase transitions described above. Indeed, it is an underlying assumption of electron microscopy experiments on aqueous systems that we should more or less preserve the room-temperature structure by rapid freezing. We carried out an electron microscopy study of n-butylammonium vermiculites at CCTR Cryotech, York, U.K. [4],... 

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

An elegant example of the measurement of an electron self-exchange rate of a redox protein was reported by Dahlin et al. The copper ion of stellacyanin was removed and then replaced with either Cu or Cu. Oxidized [ Cu] stellacyanin was allowed to react with reduced [ Cu] stellacyanin for various times (10 ms to 7 min) at 20°C, after which the reaction was quenched by lowering the solution temperature to - 120°C using a rapid-freeze apparatus ... 

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. ... 

The rapid-quench method [78] was used in Ref. 83 to analyze the mechanism of a bacterial phenylalanine hydroxylase, a mononuclear nonheme iron protein that uses tetrahydropterin as the source of the two electrons needed to activate O2 for the hydroxylation of phenylalanine to tyrosine. Mossbauer spectra of samples prepared by freeze-quenching the reaction of the complex enzyme— Fe(ll)-phenylalanine-6-methyltetrahydropterin with O2 revealed the accumulation of an intermediate at short reaction times (20-100 ms). The Mossbauer parameters of the intermediate (3 = 0.28mms, A q= l.26mms ) suggested it to be a high-spin Fe(IV) complex similar to those that have previously been detected in the reactions of other mononuclear Fe(ll) hydroxylases. 

Figure lb Electron micrograph of a 20% KCl solution after rapid quenching to —150°C and freeze-drying at —70°C. Note the radial crystallisation pattern, originating from the nucleation site at the centre of the picture. Modified from MacKenzie ... 

---