A novel freeze-quench instrument with a characteristic <> of 137 +/- 18 micros 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 m s(-1)); (b) tangential micro-mixer with an operating volume of approximately 1 nl yields short mixing times (up to 20 micros); (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 (approximately 20 microm) and a high jet velocity (approximately 200 m s(-1)) provide high sample-cooling rates, resulting in a short cryofixation time (up to 30 micros). The dynamic range of the freeze-quench device is between 130 micros and 15 ms. The novel tangential micro-mixer efficiently mixes viscous aqueous solutions, showing more than 95% mixing at eta < or = 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 bo3-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 o3 in the first 137 micros 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 o3. The radical decays within approximately 1 ms concomitant with the oxidation of heme b, and can be attributed to the PM reaction intermediate converting to the oxoferryl intermediate F. The general field of application of the freeze-quench methodology is discussed.

X-ray absorption spectroscopy at the sulfur K-edge (∼2470 eV) has been applied to a series of 2Fe−2S model complexes to obtain insight into their electronic structures. Since these 2Fe−2S complexes contain both terminal thiolates and bridging sulfides, contributions to covalency from both sets of ligands can be evaluated. Importantly, the pre-edge feature of sulfide can be resolved from that of thiolate due to differences in effective nuclear charge. In our previous studies, the covalency of the metal−thiolate bond in [Fe(SR)4]- was determined. In this study, sulfide covalency is quantified for the first time on the basis of an analysis of previous X-ray photoelectron and X-ray absorption spectroscopic studies of [FeCl4]- which are then applied to the bis-μ2-sulfide compound KFeS2. With references for both sulfide and thiolate covalencies thus established for open d-shell systems, comparisons are made between thiolate and sulfide bonding. Sulfide−Fe covalency in the [Fe2S2(SR)4]2- complexes is higher than thiolate−Fe covalency, indicating extensive charge donation of the bridging sulfides. Finally, this investigation of model complexes is extended to the oxidized and reduced 2Fe−2S cluster of the Rieske protein of Paracoccus denitrificans which has terminal thiolates on one Fe center, and histidines on the other Fe center. It is determined that thiolate covalency of the Fe(III) center is the same in both the oxidized and reduced Rieske clusters and similar to that of the [Fe2S2(SR)4]2- model complexes. Further, in the fully oxidized Rieske cluster, the sulfide covalency of the ferric center containing terminal histidine ligation is ∼18% higher than the Fe(III) containing terminal thiolate ligation. This is consistent with the fact that the histidine ligands are poorer donors and supports the suggestion that the terminal histidine ligation makes a significant contribution to the higher reduction potential of the Rieske protein.

T4 DNA ligase is an Mg 2+ -dependent and ATP-dependent enzyme that seals DNA nicks in three steps: it covalently binds AMP, transadenylates the nick phosphate, and catalyses formation of the phosphodiester bond releasing AMP. In this kinetic study, we further detail the reaction mechanism, showing that the overall ligation reaction is a superimposition of two parallel processes: a ÔprocessiveÕ ligation, in which the enzyme transadenylates and seals the nick without dissociating from dsDNA, and a ÔnonprocessiveÕ ligation, in which the enzyme takes part in the abortive adenylation cycle (covalent binding of AMP, transadenylation of the nick, and dissociation). At low concentrations of ATP (< 10 lM) and when the DNA nick is sealed with mismatching base pairs (e.g. five adjacent), this superimposition resolves into two kinetic phases, a burst ligation (% 0.2 min )1 ) and a subsequent slow ligation (% 2 · 10 )3 min )1 ). The relative rate and extent of each phase depend on the concentrations of ATP and Mg 2+ . The activation energies of self-adenylation (16.2 kcalAEmol )1 ), transadenylation of the nick (0.9 kcalAEmol )1 ), and nicksealing (16.3-18.8 kcalAEmol )1 ) were determined for several DNA substrates. The low activation energy of transadenylation implies that the transfer of AMP to the terminal DNA phosphate is a spontaneous reaction, and that the T4 DNA ligase-AMP complex is a high-energy intermediate. To summarize current findings in the DNA ligation field, we delineate a kinetic mechanism of T4 DNA ligase catalysis.