Peter E. Doan

Purified particulate methane monooxygenase from Methylococcus capsulatus (Bath) is a dimer with both mononuclear copper and a copper-containing cluster

Particulate methane monooxygenase (pMMO) is a membrane-bound enzyme that catalyzes the oxidation of methane to methanol in methanotropic bacteria. Understanding how this enzyme hydroxylates methane at ambient temperature and pressure is of fundamental chemical and potential commercial importance. Difficulties in solubilizing and purifying active pMMO have led to conflicting reports regarding its biochemical and biophysical properties, however. We have purified pMMO from Methylococcus capsulatus (Bath) and detected activity. The purified enzyme has a molecular mass of ≈200 kDa, probably corresponding to an α2β2γ2 polypeptide arrangement. Each 200-kDa pMMO complex contains 4.8 ± 0.8 copper ions and 1.5 ± 0.7 iron ions. Electron paramagnetic resonance spectroscopic parameters corresponding to 40–60% of the total copper are consistent with the presence of a mononuclear type 2 copper site. X-ray absorption near edge spectra indicate that purified pMMO is a mixture of Cu(I) and Cu(II) oxidation states. Finally, extended x-ray absorption fine structure data are best fit with oxygen/nitrogen ligands and a 2.57-Å Cu-Cu interaction, providing direct evidence for a copper-containing cluster in pMMO.

Internal dynamics of a supramolecular nanofibre

A large variety of functional self-assembled supramolecular nanostructures have been reported over recent decades.1 The experimental approach to these systems initially focused on the design of molecules for specific interactions that lead to discrete geometric structures.1–4 Recently, kinetics and mechanistic pathways of self-assembly have been investigated,6,7 but there remains a major gap in our understanding of internal conformational dynamics and their links to function. This challenge has been addressed through computational chemistry with the introduction of molecular dynamics (MD) simulations, which yield information on molecular fluctuations over time.5–7 Experimentally, it has been difficult to obtain analogous data with sub-nanometer spatial resolution. Thus, there is a need for experimental dynamics measurements, to confirm and guide computational efforts and to gain insight into the internal motion in supramolecular assemblies. Using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy, we measured conformational dynamics through the 6.7 nm cross-section of a self-assembled nanofiber in water and provide unique insight for the design of supramolecular functional materials.

Metalloenzyme Active-Site Structure and Function through Multifrequency CW and Pulsed ENDOR

Electron paramagnetic resonance (EPR) techniques have long been major tools in efforts to determine the structure and function of metalloen-zyme active sites (Beinert et al., 1962; Hoff, 1989). Much of the information EPR provides about the composition, structure, and bonding of a paramagnetic metal center is obtained by analysis of hyperfine coupling constants (Abragam and Bleaney, 1970; Atherton, 1973) that arise from interactions between the spin of the unpaired electron(s) and the spins of nuclei associated with the metal center, endogenous ligands, or bound substrate. At the most basic level, the observation of hyperfine coupling and its assignment to one or more nuclei (e.g., 1H, 14N) provide information about the chemical composition of the center. Detailed analysis of these couplings can provide information about its geometry or about substrate binding, as well as deep insights into chemical bonding. In principle these coupling constants can be calculated from splittings in the EPR spectrum. However, as illustrated in Fig. 1, for most metalloproteins these splittings cannot be resolved, and thus the chemical information they carry is lost.

Making hyperfine selection in Mims ENDOR independent of deadtime

Abstract A form of remote echo-detected ENDOR can be performed by adding an additional π pulse to the detection phase of a Mims three-pulse, stimulated-echo ENDOR experiment. The resulting four-pulse experiment, denoted refocused Mims (ReMims) ENDOR, retains the hyperfine selectivity of the Mims technique, but makes it possible to obtain arbitrarily short preparation intervals, τ 1 , independent of the spectrometer deadtime. As a result, without resonator redesign, it is possible to study a wider range of hyperfine values without distortions from “blind-spots”. The hyperfine selectivity of the ReMims ENDOR sequence is demonstrated on the 1 H and 14 N ENDOR spectra from a single crystal of Cu(glycine) 2 . Use of the ReMims protocol to augment the basic Mims and Davies ENDOR sequences is shown in two different types of situation: the sign of a 9 MHz coupled proton is assigned by the use of the implicit-TRIPLE effect in the proton ENDOR spectrum of nitrile hydratase; the true lineshape of the proton ENDOR spectrum of fluorometmyoglobin is determined without distortions by blindspots.

Quantitative studies of davies pulsed ENDOR

Abstract The results of quantitative and systematic studies of the dependence of two-pulse spin-echo intensity on the pulse widths and of the Davies ENDOR response on the width of the preparation pulse, t p are reported. A formula that describes the Davies ENDOR response as a function of the selectivity parameter, η η = A n t p is presented. These measurements, which employ a variety of small molecules and metalloenzymes, not only provide information about the Davies ENDOR technique, but also give a quantitative description of the recently described POSHE method for hyperfine selection of heteronuclear pulsed ENDOR spectra through manipulation of pulse widths in the Davies ENDOR sequence. This procedure involves suppression of weakly coupled proton pattern and optimization of strongly coupled heteronuclei signals. It is demonstrated with single-crystal data for Cu(II)-doped Zn(glycinato) 2 and frozen solution data for the blue copper protein Pseudomonas aeruginosa azurin.

Identification of protonated oxygenic ligands of ribonucleotide reductase intermediate X.

We previously used a combination of continuous-wave (CW) and pulsed electron-nuclear double resonance (ENDOR) protocols to identify the types of protonated oxygen (OH(x)) species and their disposition within the Fe(III)/Fe(IV) cluster of intermediate X, the direct precursor of the essential diferric-tyrosyl radical cofactor of the beta2 subunit of Escherichia coli ribonucleotide reductase (RNR). We concluded that X contains the [(H(x)O)Fe(III)OFe(IV)] fragment (T model), and does not contain a mu-hydroxo bridge. When combined with a subsequent (17)O ENDOR study of X prepared with H(2)(17)O and with (17)O(2), the results led us to suggest that this fragment is the entire inorganic core of X. This has been questioned by recent reports, but these reports do not themselves agree on the core of X. One concluded that X possesses a di-mu-oxo Fe(III)/Fe(IV) core plus a terminal (H(2)O) bound to Fe(III) [e.g., Han, W.-G.; Liu, T.; Lovell, T.; Noodleman, L. J. Am. Chem. Soc. 2005, 127, 15778-15790]. The other [Mitic, N.; Clay, M. D.; Saleh, L.; Bollinger, J. M.; Solomon, E. I. J. Am. Chem. Soc. 2007, 129, 9049-9065] concluded that X contains only a single oxo bridge and postulated the presence of an additional hydroxo bridge plus a terminal hydroxyl bound to Fe(III). In this report we take advantage of improvements in 35 GHz pulsed ENDOR performance to reexamine the protonation state of oxygenic ligands of the inorganic core of X by directly probing the exchangeable proton(s) with (2)H pulsed ENDOR spectroscopy. These (2)H ENDOR measurements confirm that X contains an Fe(III)-bound terminal aqua ligand (H(x)O), but the spectra contain none of the features that would be required for the proton of a bridging hydroxyl. Thus, we confirm that X contains a terminal aqua (most likely hydroxo) ligand to Fe(III) in addition to one or two mu-oxo bridges but does not contain a mu-hydroxo bridge. The (2)H ENDOR measurements further demonstrate that this conclusion is applicable to both wild type and Y122F-beta2 mutant, and in fact we detect no difference between the properties of protons on the terminal oxygens in the two variants; likewise, (14)N ENDOR measurements of histidyl ligands bound to Fe show no difference between the two variants.

A simple method for hyperfine-selective heteronuclear pulsed ENDOR via proton suppression

A major reason for the importance of ENDOR spectroscopy ( 1) in the study of metal complexes is that the technique is inherently broad-banded: all coupled nuclei having spin I > 0 can be detected with comparable sensitivity (2). Unfortunately, at X-band magnetic fields ( -0.3 T at g = 2) the proton ENDOR spectrum often overlaps with and obscures the spectra of important heteronuclei such as 14N, 13C 57Fe or 33S. One solution is to work at higher microwave frequency (2, 3). In adiitioi, three elegant, two-dimensional pulsed-ENDOR techniques (4) have been developed to address this problem by hyperfine selection at X band (57). We now describe a simple one-dimensional pulsed-ENDOR method for hyperfine selection of heteronuclear ENDOR signals through the suppression of the proton pattern. As the technique also leads to enhancement of the intensities of heteronuclear signals, we call it POSHEENDOR (proton suppression, heteronuclear enhancement). Two of the currently available pulsed-ENDOR techniques for hyperiine selection are formally equivalent and can be loosely grouped as ELDOR-ENDOR experiments. Buhlmann et al. (5) used field jumps within a Davies ENDOR sequence (4, 8) to achieve selection, whereas Thomann and Bernard0 (6) used microwave frequency jumps. However, these methods require two-dimensional experiments to obtain a complete hyperfme-selected spectrum and thus need large blocks of time for data acquisition. In addition, they require equipment beyond that necessary for ordinary Davies ENDOR. The third technique, developed by de Beer et al. ( 7)) utilizes a Mims ENDOR sequence (4, 9). In this method, hyperfine selection is achieved by systematic variation of time between the first and second microwave pulses in a stimulated-echo sequence. This procedure is restricted by the time required for a 2D experiment as well as by limitation of the Mims sequence to smaller hypetine couplings. The hyperfine selection technique we describe employs the fact that in a Davies ENDOR sequence,

The ups and downs of Feher-style ENDOR

A new transient variation of the “Feher-style” electron-nuclear double resonance (ENDOR) method is examined. In this technique, the passage-mode electron paramagnetic resonance (EPR) signal is monitored following the application of a pulsed radio frequency (RF). Continuous-wave and transient proton ENDOR experiments have been conducted on the nonheme iron center from the protein nitrile hydratase. These experiments show that the transient ENDOR signal response exhibits a complex response with multiple phases in the time evolution of the ENDOR signal. Both increases and decreases in the passage-mode EPR signals are observed at different times following the RF pulse that induces an ENDOR transition. A simple model based on a packet-shifting ENDOR mechanism for a nonadiabatic passage EPR signal is proposed. This model describes many of the features seen in the transient ENDOR experiments and provides new insight into the traditional Feher-style ENDOR measurements. This new model shows that a packet-shifting mechanism can account for many of the “negative ENDOR” effects commonly seen in Feher-style ENDOR, which suggests that more exotic ENDOR mechanisms may not be required to explain these observations.

How important is parallel-mode EPR in electron spin echo envelope modulation studies of non-Kramers systems?

Abstract Integer-spin systems with S >1 sometimes exhibit a ground-state ‘non-Kramers (NK) doublet’ in their electron paramagnetic resonance (EPR) spectra. The preferred method of studying systems with this type of EPR signal has been to use a spectrometer in which the microwave field is parallel to the applied static field (parallel-mode EPR), rather than a traditional perpendicular-mode EPR spectrometer, in order to maximize the resulting NK-EPR signal. The efficacy of parallel and perpendicular mode electron spin echo envelope modulation (ESEEM) spectroscopy on the NK doublet of azido-hemerythrin (N 3 Hr red ) are compared. These results demonstrate that for this technique, the advantages of parallel-mode over perpendicular-mode ESEEM are minimal at best. A simplified form of the theory underlying the analysis of NK-ESEEM is developed to explain the observations.