Rapid Freeze-quench Research Articles

Evaluation of Copper(II) Transfer between Amyloid-beta Peptides by Relaxation-Induced Dipolar Modulation Enhancement (RIDME).

In the brains of Alzheimer's disease patients, fibrillar aggregates containing amyloid-beta (Aβ) peptides are found, along with elevated concentrations of Cu(II) ions. The aggregation pathways of Aβ peptides can be modulated by Cu(II) ions and is determined by the formation and nature of the Cu(II)-Aβ complex. If spin-labeled, the Cu(II)-Aβ complex contains two dipolar coupled paramagnetic centers, the spin label and the Cu(II) ion. Measurement of the dipolar coupling between these paramagnetic centers by relaxation-induced dipolar modulation enhancement (RIDME) allows to monitor the complex formation and thus opens a way to follow the Cu(II) transfer between peptides if a mixture of wild-type and spin-labeled ones is used. We evaluate this approach for a specific Cu(II)-Aβ complex, the aggregation-inert Component II. The kinetics of the Cu(II) transfer can be resolved by performing RIDME in a time-dependent manner. A temporal resolution of seconds has been achieved, with the potential to reach milliseconds, using a rapid-freeze quench device to stop the Cu(II) transfer in solution after defined incubation times.

Intramolecular electron transfer from biopterin to FeII-O2 complex in nitric oxide synthases occurs at very different rates between bacterial and mammalian enzymes: Direct observation of a catalytically active intermediate

Nitric oxide synthase (NOS) is a cytochrome P450-type mono‑oxygenase that catalyzes the oxidation of L-arginine to nitric oxide. We previously observed that intramolecular electron transfer from biopterin to Fe2+-O2 in Deinococcus radiodurans NOS (DrNOS) using pulse radiolysis. However, the rate of electron transfer in DrNOS (2.2 × 103 s−1) contrasts with a reported corresponding rate (11 s−1) in a mammalian NOS determined using rapid freeze-quench (RFQ) EPR. We applied pulse radiolysis to Bacillus subtilis NOS (bsNOS) and to rat neural NOS oxygenase domain NOS (mNOS). Concurrently, RFQ EPR was used to trap a pterin radical during single-turnover enzyme reactions of the enzymes. By using the pulse radiolysis method, hydrated electrons (eaq−) reduced the heme iron of NOS enzymes. Subsequently, ferrous heme reacted with O2 to form a Fe2+-O2 intermediate. In the presence of pterin, the intermediate of bsNOS was found to convert to other intermediate in the time range of milliseconds. A similar process was determined to have occurred after pulse radiolysis of the pterin-bound mNOS, though the rate was much slower. The intermediates of all of the NOS enzymes further converted to the original ferric form in the time range of seconds. When using the RFQ method, pterin radicals were formed very rapidly in both DrNOS and bsNOS in the time range of milliseconds. In contrast, the pterin radical in mNOS was observed to form slowly, at a rate of ∼20 s−1.

Pre-Steady-State Reactivity of Peptidylglycine Monooxygenase Implicates Ascorbate in Substrate Triggering of the Active Conformer.

Peptidylglycine monooxygenase (PHM) is essential for the posttranslational amidation of neuroendocrine peptides. An important aspect of the PHM mechanism is the complete coupling of oxygen reduction to substrate hydroxylation, which implies no oxygen reactivity of the fully reduced enzyme in the absence of peptidyl substrates. As part of studies aimed at investigating this feature of the PHM mechanism, we explored pre-steady-state kinetics using chemical quench (CQ) and rapid freeze-quench (RFQ) studies of the fully reduced ascorbate-free PHM enzyme. First, we confirmed the absence of Cu(I)-enzyme oxidation by O2 at catalytic rates in the absence of peptidyl substrate. Next, we investigated reactivity in the presence of the substrate dansyl-YVG. Surprisingly, when ascorbate-free di-Cu(I) PHM was shot against oxygenated buffer containing the dansyl-YVG substrate, <15% of the expected product was formed. Substoichiometric reactivity was confirmed by stopped-flow and RFQ EPR spectroscopy. Product generation reached a maximum of 70% by the addition of increasing amounts of the ascorbate cosubstrate in a process that was not the result of multiple turnovers. FTIR spectroscopy of the Cu(I)-CO reaction chemistry was then used to show that increasing ascorbate concentrations correlated with a substrate-induced Cu(I)M-CO species characteristic of an altered conformation. We conclude that ascorbate and peptidyl substrate work together to induce a transition from an inactive to an active conformation and suggest that the latter may represent the "closed" conformation (Cu-Cu of ∼4 Å) recently observed for both PHM and its sister enzyme DBM by crystallography.

Recent progress in radical SAM enzymes: New reactions and mechanisms

<p indent="0mm"><italic>S</italic>-adenosyl-<italic>L</italic>-methionine (SAM) is the second largest cofactor in the human body, and its metabolism is closely related to various physiological activities. In addition to a methyl donor, SAM is also used by radical SAM enzymes with a [4Fe-4S] cluster to catalyze a series of radical reactions. Since the discovery of this enzyme family, it has been found to play important physiological functions in all kingdoms of life. According to bioinformatics prediction, over 220000 radical SAM enzymes are involved in more than 85 biochemical transformations. Almost all radical SAM enzymes have a conserved CxxxCxxC motif, which coordinates the [4Fe-4S] cluster. The reduced [4Fe-4S] cluster provides an electron to SAM to cleave the C_{5′,adenosine}−S bond of SAM and generates a 5′-deoxyadenosine radical (5′-dA·). This radical then grabs a hydrogen atom from the substrate to generate the substrate radical and initiates many different types of reactions. These include thioether crosslinking reactions, radical addition reactions that generate C–C bonds, aliphatic etherification that generates C−O bonds, oxidation reactions, complex rearrangement reactions, methylation, methylthiolation, cyclopropanation reactions and so on. The nonclassical radical SAM enzyme Dph2 cleaves the C_γ,Met–S bond of SAM to generate a 3-amino-3-carboxylpropyl radical (ACP radical), which is added to a histidine residue of the substrate protein elongation factor EF2 for diphthamide biosynthesis. Radical SAM enzymes have a very rich substrate scope, including ribosomally synthesized and posttranslationally modified peptides (RiPPs), proteins, nucleosides and all kinds of small molecules. Classifying by the type of substrate, this review summarizes some of the newly discovered radical SAM enzymes since 2015 and introduces the catalytic mechanism. In addition to new enzymes and reactions, strides have been made in mechanistic studies of radical SAM enzymes in the past several years. Using rapid freeze-quench (RFQ) combined with electron spin resonance spectroscopy (EPR) and electron-nucleus double resonance spectroscopy (ENDOR), researchers have captured and characterized two types of novel intermediates in radical SAM enzymes. These two intermediates are organometallic species with Fe–C bonds formed by the iron-sulfur cluster with the 5′-dA radical and ACP radical, respectively. These findings answer the long-standing question of how enzymes control active organic radical species in radical SAM enzymes and the regioselectivity of SAM cleavage. Finally, we look forward to the future directions of radical SAM enzymes. As discussed above, radical SAM enzymes catalyze numerous difficult reactions, such as C−H activation and C−C bond formation. New enzymatic reactions with unnatural substrates could be developed with protein engineering. Therefore, natural product analogs could be easily made for drug development. Furthermore, in the radical SAM chemistry, only Dph2 can cleave SAM unconventionally to generate the ACP radical, other than the 5′-dA radical from all the other radical SAM enzymes. Whether there are more enzymes using this mechanism to catalyze the reaction remains unexplored. We expect that more Dph2-like enzymes that can generate ACP radicals will be discovered in the future. Finally, the drawback for almost all the reported radical SAM enzymes thus far is their low efficiency. Protein engineering or other strategies that could increase the stability and efficiency of radical SAM enzymes are in high demand. Only by this means would we expect more widespread use of radical SAM enzymes as biocatalysts in synthetic biology.

A New Reaction for Improved Calibration of EPR Rapid-Freeze Quench Times: Kinetics of Ethylene Diamine Tetraacetate (EDTA) Transfer from Calcium(II) to Copper(II).

The kinetics of the transfer of the chelate, ethylenediamine tetraacetate (EDTA), from Calcium(II) to Copper(II) in imidazole (Im) buffers near neutral pH, corresponding to the conversion, [Cu(II)Im4]2+→ [Cu(II)EDTA]2-, are characterized with stopped-flow absorption spectroscopy and implemented as a tool for calibrating the interval between mixing and freezing, the freeze-quench time (t Q ), of a rapid freeze-quench (RFQ) apparatus. The kinetics of this reaction are characterized by monitoring changes in UV-visible spectra (300 nm) due to changes in the charge-transfer band associated with the Cu2+ ions upon EDTA binding. Stopped-flow measurements show that the rates of conversion of the Cu2+ ions exhibit exponential kinetics on millisecond time scales at pH values less than 6.8. In parallel, we have developed a simple but precise method to quantitate the speciation of frozen solution mixtures of [Cu(II)(EDTA)]2- and tetraimidazole Cu(II) ([Cu(Im)4]2+) in X-band EPR spectra. The results are implemented in a simple high-precision 'recipe' for determining t Q . These procedures are more accurate and precise than the venerable reaction of aquometmyoglobin with azide for calibrating RFQ apparatus, with the benefit of avoiding high-concentrations of toxic azide solutions.

Millisecond Time-Resolved Solid-State NMR Reveals a Two-Stage Molecular Mechanism for Formation of Complexes between Calmodulin and a Target Peptide from Myosin Light Chain Kinase.

Calmodulin (CaM) mediates a wide range of biological responses to changes in intracellular Ca2+ concentrations through its calcium-dependent binding affinities to numerous target proteins. Binding of two Ca2+ ions to each of the two four-helix-bundle domains of CaM results in major conformational changes that create a potential binding site for the CaM binding domain of a target protein, which also undergoes major conformational changes to form the complex with CaM. Details of the molecular mechanism of complex formation are not well established, despite numerous structural, spectroscopic, thermodynamic, and kinetic studies. Here, we report a study of the process by which the 26-residue peptide M13, which represents the CaM binding domain of skeletal muscle myosin light chain kinase, forms a complex with CaM in the presence of excess Ca2+ on the millisecond time scale. Our experiments use a combination of selective 13C labeling of CaM and M13, rapid mixing of CaM solutions with M13/Ca2+ solutions, rapid freeze-quenching of the mixed solutions, and low-temperature solid state nuclear magnetic resonance (ssNMR) enhanced by dynamic nuclear polarization. From measurements of the dependence of 2D 13C-13C ssNMR spectra on the time between mixing and freezing, we find that the N-terminal portion of M13 converts from a conformationally disordered state to an α-helix and develops contacts with the C-terminal domain of CaM in about 2 ms. The C-terminal portion of M13 becomes α-helical and develops contacts with the N-terminal domain of CaM more slowly, in about 8 ms. The level of structural order in the CaM/M13/Ca2+ complexes, indicated by 13C ssNMR line widths, continues to increase beyond 27 ms.

Kinetic and Spectroscopic Characterization of the Catalytic Ternary Complex of Tryptophan 2,3-Dioxygenase.

The first step of the kynurenine pathway for l-tryptophan (l-Trp) degradation is catalyzed by heme-dependent dioxygenases, tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase. In this work, we employed stopped-flow optical absorption spectroscopy to study the kinetic behavior of the Michaelis complex of Cupriavidus metallidurans TDO (cmTDO) to improve our understanding of oxygen activation and initial oxidation of l-Trp. On the basis of the stopped-flow results, rapid freeze-quench (RFQ) experiments were performed to capture and characterize this intermediate by Mössbauer spectroscopy. By incorporating the chlorite dismutase-chlorite system to produce high concentrations of solubilized O2, we were able to capture the Michaelis complex of cmTDO in a nearly quantitative yield. The RFQ-Mössbauer results confirmed the identity of the Michaelis complex as an O2-bound ferrous species. They revealed remarkable similarities between the electronic properties of the Michaelis complex and those of the O2 adduct of myoglobin. We also found that the decay of this reactive intermediate is the rate-limiting step of the catalytic reaction. An inverse α-secondary substrate kinetic isotope effect was observed with a kH/kD of 0.87 ± 0.03 when (indole-d5)-l-Trp was employed as the substrate. This work provides an important piece of spectroscopic evidence of the chemical identity of the Michaelis complex of bacterial TDO.

The Catalytic Role of a Conserved Tyrosine in Nitric Oxide-Reducing Non-heme Diiron Enzymes

Recent studies have identified several key intermediates of the nitric oxide reductase (NOR) cycle of flavodiiron proteins (FDPs). These intermediates include, sequentially, a μ-hydroxo diferrous species, a mono-NO intermediate, a di-NO intermediate (FDPdiNO), and a bis-μ-hydroxo diferric species. This paper focuses on the reaction path connecting the last two intermediates on which the nitrous oxide product is released. On the basis of density functional theory calculations, a reaction sequence is proposed in which the enzyme passes through an intermediate with a bridging hyponitrite ligand, which accepts a proton, initiating a rate-determining ligand rotation from an N/N- to an N/O-coordinated conformation. This rotation is facilitated by a second-sphere tyrosine residue, which provides a transient hydrogen bond to one of the nitrogen atoms of the substrate near the transition state. The role of the tyrosine residue in the NOR activity has been tested by steady-state kinetics and rapid freeze-quench (RFQ) studies of the Y197F variant of the FDP from T. maritima in which the hydrogen bonding interaction is absent. The Y197F variant displayed little or no steady-state NOR activity in support of the importance of Y197. The RFQ samples, monitored by Mössbauer spectroscopy, showed that Y197F follows the same reaction path as a wild-type FDP up to and including the formation of FDPdiNO but diverges subsequently with the variant forming an inactive mono-NO species. The RFQ results demonstrate that Y197 enables the postFDPdiNO section of the reaction cycle in the wild-type FDP to proceed to N2O. The proposed refinement of the reaction mechanism provides an explanation for the lack of NOR activity of the variant Y197F of T. maritima and of other di-NO binding diiron enzymes and model compounds with active site structures like those of FDPs. The reported reductions of NO to N2O catalyzed by synthetic diiron complexes proceed only with the support of radiation, additional electrons, or an electron-rich ligand environment. However, higher potential diiron sites like those of FDPs require hydrogen bonding to second-sphere residues to turn over NO to N2O.

Kinetic analysis of amino acid radicals formed in H2O2-driven CuI LPMO reoxidation implicates dominant homolytic reactivity

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with both [Formula: see text] and [Formula: see text] as cosubstrates. In this study, the [Formula: see text] reaction with reduced Hypocrea jecorina LPMO9A (CuI-HjLPMO9A) is demonstrated to be 1,000-fold faster than the [Formula: see text] reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze-quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce CuII-HjLPMO9A and indicates homolytic O-O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the CuII sites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism.

Rates and equilibrium constants of the ligand-induced conformational transition of an HCN ion channel protein domain determined by DEER spectroscopy.

Ligand binding can induce significant conformational changes in proteins. The mechanism of this process couples equilibria associated with the ligand binding event and the conformational change. Here we show that by combining the application of W-band double electron-electron resonance (DEER) spectroscopy with microfluidic rapid freeze quench (μRFQ) it is possible to resolve these processes and obtain both equilibrium constants and reaction rates. We studied the conformational transition of the nitroxide labeled, isolated carboxy-terminal cyclic-nucleotide binding domain (CNBD) of the HCN2 ion channel upon binding of the ligand 3',5'-cyclic adenosine monophosphate (cAMP). Using model-based global analysis, the time-resolved data of the μRFQ DEER experiments directly provide fractional populations of the open and closed conformations as a function of time. We modeled the ligand-induced conformational change in the protein using a four-state model: apo/open (AO), apo/closed (AC), bound/open (BO), bound/closed (BC). These species interconvert according to AC + L ⇌ AO + L ⇌ BO ⇌ BC. By analyzing the concentration dependence of the relative contributions of the closed and open conformations at equilibrium, we estimated the equilibrium constants for the two conformational equilibria and the open-state ligand dissociation constant. Analysis of the time-resolved μRFQ DEER data gave estimates for the intrinsic rates of ligand binding and unbinding as well as the rates of the conformational change. This demonstrates that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational change.

Rapid Freeze-Quench EPR Spectroscopy: Improved Collection of Frozen Particles.

Rapid freeze-quench (RFQ) in combination with electron paramagnetic resonance (EPR) spectroscopy at X-band is a proven technique to trap and characterize paramagnetic intermediates of biochemical reactions. Preparation of suitable samples is still cumbersome, despite many attempts to remedy this problem, and limits the wide applicability of RFQ EPR. We present a method, which improves the collection of freeze-quench particles from isopentane and their packing in an EPR tube. The method is based on sucking the particle suspension into an EPR tube with a filter at the bottom. This procedure results in a significant reduction of the required volume of reactants, which allows the economical use of valuable reactants such as proteins. The approach also enables the successful collection of smaller frozen particles, which are generated at higher flow rates. The method provides for a reproducible, efficient and fast collection of the freeze-quench particles and can be easily adapted to RFQ EPR at higher microwave frequencies than X-band.

A Microfluidic Rapid Freeze Quench Apparatus for High Field EPR Measurements

Rapid freeze quench (RFQ) EPR is a well-established technique for trapping reaction intermediates. A major difficulty in using commercial RFQ-EPR combined with standard X-band EPR spectroscopy is the relatively large amount of sample needed for each time point, and the associated amount that is wasted in the dead volume of the tubes and mixer. This is particularly prohibitive when one would like to couple RFQ with high resolution EPR techniques such as ENDOR (electron-nuclear double resonance) and DEER (electron-electron double resonance) that provide electron-nuclear and electron-electron distances and are less sensitive than standard continuous wave EPR. We have developed a dedicated microfluidic RFQ (RFQ) apparatus for W-band measurements, optimized for the small W-band sample size and a minimal sample amount for a series of ∼7 time points collected in triplicates (∼200 l of 0.03-0.1 mM labeled protein). The mixer is based on a recent published design[1] with a modified sample ejection system and cold trap. It current time window is 5-90 msec and its performance has been demonstrated on the reduction of nitroxide with dithionite[2]. The current state of the RFQ apparatus is demonstrated on : (i) the ATPase activity of an RNA helicase. Here Mg2+ was substituted with Mn2+ and the 31P ENDOR spectrum was recorded. The ADP and ATP spectra have significantly different lineshapes and can therefore be used to probe the hydrolysis state. (ii) Conformational changes induced in a protein through ligand binding as detected by DEER. 1. Egawa T, Durand JL, Hayden EY, Rousseau DL, Yeh S-R. Anal Chem. 2009;81(4):1622-7. 2. R. Kaufmann, D. Goldfarb.. J Magn Reson. 2013;230:220-6.

A Rapid Freeze‐Quench Setup for Multi‐Frequency EPR Spectroscopy of Enzymatic Reactions

Electron paramagnetic resonance (EPR) spectroscopy in combination with the rapid freeze-quench (RFQ) technique is a well-established method to trap and characterize intermediates in chemical or enzymatic reactions at the millisecond or even shorter time scales. The method is particularly powerful for mechanistic studies of enzymatic reactions when combined with high-frequency EPR (ν≥90 GHz), which permits the identification of substrate or protein radical intermediates by their electronic g values. In this work, we describe a new custom-designed micro-mix rapid freeze-quench apparatus, for which reagent volumes for biological samples as small as 20 μL are required. The apparatus was implemented with homemade sample collectors appropriate for 9, 34, and 94 GHz EPR capillaries (4, 2, and 0.87 mm outer diameter, respectively) and the performance was evaluated. We demonstrate the application potential of the RFQ apparatus by following the enzymatic reaction of PpoA, a fungal dioxygenase producing hydro(pero)xylated fatty acids. The larger spectral resolution at 94 GHz allows the discernment of structural changes in the EPR spectra, which are not detectable in the same samples at the standard 9 GHz frequency.

Solution and Solid State NMR Approaches To Draw Iron Pathways in the Ferritin Nanocage

Ferritins are intracellular proteins that can store thousands of iron(III) ions as a solid mineral. These structures autoassemble from four-helix bundle subunits to form a hollow sphere and are a prototypical example of protein nanocages. The protein acts as a reservoir, encapsulating iron as ferric oxide in its central cavity in a nontoxic and bioavailable form. Scientists have long known the structural details of the protein shell, owing to very high resolution X-ray structures of the apoform. However, the atomic level mechanism governing the multistep biomineralization process remained largely elusive. Through analysis of the chemical behavior of ferritin mutants, chemists have found the role of some residues in key reaction steps. Using Mössbauer and XAS, they have identified some di-iron intermediates of the catalytic reaction trapped by rapid freeze quench. However, structural information about the iron interaction sites remains scarce. The entire process is governed by a number of specific, but weak, interactions between the protein shell and the iron species moving across the cage. While this situation may constitute a major problem for crystallography, NMR spectroscopy represents an optimal tool to detect and characterize transient species involving soluble proteins. Regardless, NMR analysis of the 480 kDa ferritin represents a real challenge. Our interest in ferritin chemistry inspired us to use an original combination of solution and solid state approaches. While the highly symmetric structure of the homo-24-mer frog ferritin greatly simplifies the spectra, the large protein size hinders the efficient coherence transfer in solution, thus preventing the sequence specific assignments. In contrast, extensive (13)C-spin diffusion makes the solution (13)C-(13)C NOESY experiment our gold standard to monitor protein side chains both in the apoprotein alone and in its interaction with paramagnetic iron species, inducing line broadening on the resonances of nearby residues. We could retrieve the structural information embedded in the (13)C-(13)C NOESY due to a partial sequence specific assignment of protein backbone and side chains we obtained from solid state MAS NMR of ferritin microcrystals. We used the 59 assigned amino acids (∼33% of the total) as probes to locate paramagnetic ferric species in the protein cage. Through this approach, we could identify ferric dimers at the ferroxidase site and on their pathway towards the nanocage. Comparison with existing data on bacterioferritins and bacterial ferritins, as well as with eukaryotic ferritins loaded with various nonfunctional divalent ions, allowed us to reinterpret the available information. The resulting picture of the ferroxidase site is slightly different with various ferritins but is designed to provide multiple and generally weak iron ligands. The latter assist binding of two incoming iron(II) ions in two proximal positions to facilitate coupling with oxygen. Subsequent oxidation is accompanied by a decrease in the metal-metal distance (consistent with XAS/Mössbauer) and in the number of protein residues involved in metal coordination, facilitating the release of products as di-iron clusters under the effect of new incoming iron(II) ions.

NMR at low and ultralow temperatures.

Solid state nuclear magnetic resonance (NMR) measurements at low temperatures have been common in physical sciences for many years and are becoming increasingly important in studies of biomolecular systems. This Account reviews a diverse set of projects from my laboratory, dating back to the early 1990s, that illustrate the motivations for low-temperature solid state NMR, the types of information that are available from the measurements, and likely directions for future research. These projects include NMR studies of both physical and biological systems, performed at low (cooled with nitrogen, down to 77 K) and ultralow (cooled with helium, below 77 K) temperatures, and performed with and without magic-angle spinning (MAS). NMR studies of physical systems often focus on phenomena that occur only at low temperatures. Two examples from my laboratory are studies of molecular rotation and orientational ordering in solid C60 at low temperatures and studies of unusual electronic states, called skyrmions, in two-dimensionally confined electron systems within semiconductor quantum wells. To study quantum wells, we used optical pumping of nuclear spin polarizations to enhance their NMR signals. The optical pumping phenomenon exists only at ultralow temperatures. In studies of biomolecular systems, low-temperature NMR has several motivations. In some cases, low temperatures suppress molecular tumbling, thereby permitting solid state NMR measurements on soluble proteins. Studies of AIDS-related peptide/antibody complexes illustrate this effect. In other cases, low temperatures suppress conformational exchange, thereby permitting quantitation of conformational distributions. Studies of chemically denatured states of the model protein HP35 illustrate this effect. Low temperatures and rapid freeze-quenching can also be used to trap transient intermediate states in a non-equilibrium kinetic process, as shown in studies of a transient intermediate in the rapid folding pathway of HP35. NMR sensitivity generally increases with decreasing sample temperature. Therefore, it can be useful to carry out experiments at the lowest possible temperatures, particularly in studies of biomolecular systems in frozen solutions. However, solid state NMR studies of biomolecular systems generally require rapid MAS. A novel MAS NMR probe design that uses nitrogen gas for sample spinning and cold helium only for sample cooling allows a wide variety of solid state NMR measurements to be performed on biomolecular systems at 20-25 K, where signals are enhanced by factors of 12-15 relative to measurements at room temperature. MAS NMR at ultralow temperatures also facilitates dynamic nuclear polarization (DNP), allowing sizeable additional signal enhancements and large absolute NMR signal amplitudes with relatively low microwave powers. Current research in my laboratory seeks to develop and exploit DNP-enhanced MAS NMR at ultralow temperatures, for example, in studies of transient intermediates in protein folding and aggregation processes and studies of peptide/protein complexes that can be prepared only at low concentrations.

Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase.

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(•)) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(•) ⇆ [W(48)?] ⇆ Y(356) within β to Y(731) ⇆ Y(730) ⇆ C(439) within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO(2)Y(•); 1.2 radicals/β2) in place of Y(122)(•) in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y(•) proposed to be located on Y(356) in β2. We now report three independent methods that establish that Y(356) is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y(731) and Y(730) in α2. Pulsed electron-electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO(2)Y(122)(•)-Y(356)(•), NO(2)Y(122)(•)-NO(2)Y(122)(•), and NO(2)Y(122)(•)-Y(731(730))(•), respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y(731)(3,5-F(2)Y) or Y(730)(3,5-F(2)Y)-α2, which revealed F(2)Y(•), studies using globally incorporated [β-(2)H(2)]Y-α2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y(•) delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y(•) radicals in the PCET pathway and their conformations.

Investigating the intermediates in the reaction of ribonucleoside triphosphate reductase from Lactobacillus leichmannii: An application of HF EPR–RFQ technology

In this investigation high-frequency electron paramagnetic resonance spectroscopy (HFEPR) in conjunction with innovative rapid freeze-quench (RFQ) technology is employed to study the exchange-coupled thiyl radical–cob(II)alamin system in ribonucleotide reductase from a prokaryote Lactobacillus leichmannii. The size of the exchange coupling ( J ex) and the values of the thiyl radical g tensor are refined, while confirming the previously determined (Gerfen et al. (1996) [20]) distance between the paramagnets. Conclusions relevant to ribonucleotide reductase catalysis and the architecture of the active site are presented. A key part of this work has been the development of a unique RFQ apparatus for the preparation of millisecond quench time RFQ samples which can be packed into small (0.5 mm ID) sample tubes used for CW and pulsed HFEPR – lack of this ability has heretofore precluded such studies. The technology is compatible with a broad range of spectroscopic techniques and can be readily adopted by other laboratories.

Radical formation in cytochrome c oxidase

The formation of radicals in bovine cytochrome c oxidase (bC cO), during the O 2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bC cO under single turnover conditions, the reaction of O 2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 μs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO 2 − ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O 2 with reduced bC cO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H 2O 2) with oxidized bC cO was studied at pH 6 and pH 8 by trapping the products at 50 μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (R O O ) species was formed, presumably by the reaction between O 2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a 3. This article is part of a Special Issue entitled: “Allosteric cooperativity in respiratory proteins”.

Avoiding premature oxidation during the binding of Cu(II) to a dithiolate site in BsSCO. A rapid freeze-quench EPR study

The Bacillus subtilis version of SCO1 (BsSCO) is required for assembly of CuA in cytochrome c oxidase and may function in thiol-disulfide exchange and/or copper delivery. BsSCO binds Cu(II) with ligation by two cysteines, one histidine and one water. However, copper is a catalyst of cysteine oxidation and BsSCO must avoid this reaction to remain functional. Time resolved, rapid freeze-quench (RFQ) electron paramagnetic resonance of apo-BsSCO reacting with Cu(II) reveals an initial Cu(II) species with two equatorially coordinated nitrogen atoms, but no sulfur. We propose that BsSCO evolves from this initial sulfur free coordination of Cu(II) to the final dithiolate species via a change in conformation, and that the initial binding by nitrogen is a means for BsSCO to avoid premature thiol oxidation.

Design, Implementation, Simulation, and Visualization of a Highly Efficient RIM Microfluidic Mixer for Rapid Freeze-Quench of Biological Samples

Rapid freeze-quench (RFQ) trapping of short-lived reaction intermediates for spectroscopic study plays an important role in the characterization of biological reactions. Recently there has been considerable effort to achieve submillisecond reaction deadtimes. We present here a new, robust, high-velocity microfluidic mixer that enables such rapid freeze-quenching. It is a based on the mixing method of two impinging jets commonly used in reaction injection molding (RIM) of plastics. This method achieves efficient mixing by inducing chaotic flow at relatively low Reynolds numbers (Re =140). We present the first mathematical simulation and microscopic visualization of mixing in such RFQ micromixers, the results of which show that the impinging solutions efficiently mix within the mixing chamber. These tests, along with a practical demonstration in a RFQ setup that involves copper wheels, show this new mixer can in practice provide reaction deadtimes as low as 100 microseconds.