Alice M. Bowen

Structural information from orientationally selective DEER spectroscopy

Double electron-electron resonance (DEER) spectroscopy can determine, from measurement of the dipolar interaction, the distance and orientation between two paramagnetic centres in systems lacking long-range order such as powders or frozen solution samples. In spin systems with considerable anisotropy, the microwave pulses excite only a fraction of the electron paramagnetic resonance (EPR) spectrum and the resulting orientation selection needs to be explicitly taken into account if a meaningful distance and orientation is to be determined. Here, a general method is presented to analyze the dipolar interaction between two paramagnetic spin centres from a series of DEER traces recorded so that different orientations of the spin-spin vector are sampled. Delocalised spin density distributions and spin projection factors (as for example in iron-sulfur clusters), are explicitly included. Application of the analysis to a spin-labelled flavoprotein reductase/reduced iron-sulfur ferredoxin protein complex and a bi-radical with two Cu(ii) ions provides distance and orientation information between the radical centres. In the protein complex this enables the protein-protein binding geometry to be defined. Experimentally, orientationally selective DEER measurements are possible on paramagnetic systems where the resonator bandwidth allows the frequencies of pump and detection pulses to be separated sufficiently to excite enough orientations to define adequately the spin-spin vector.

Exploiting orientation-selective DEER : determining molecular structure in systems containing Cu(II) centres

Orientation-selective DEER (Double Electron-Electron Resonance) measurements were conducted on a series of rigid and flexible molecules containing Cu(ii) ions. A system with two rigidly held Cu(ii) ions was afforded by the protein homo-dimer of copper amine oxidase from Arthrobacter globiformis. This system provided experimental DEER data between two Cu(ii) ions with a well-defined distance and relative orientation to assess the accuracy of the methodology. Evaluation of orientation-selective DEER (os DEER) on systems with limited flexibility was probed using a series of porphyrin-based Cu(ii)-nitroxide and Cu(ii)-Cu(ii) model systems of well-defined lengths synthesized for this project. Density functional theory was employed to generate molecular models of the conformers for each porphyrin-based Cu(ii) dimer studied. Excellent agreement was found between DEER traces simulated using these computed conformers and the experimental data. The performance of different parameterised structural models in simulating the experimental DEER data was also investigated. The results of this analysis demonstrate the degree to which the DEER data define the relative orientation of the two Cu(ii) ions and highlight the need to choose a parameterised model that captures the essential features of the flexibility (rotational freedom) of the system being studied.

Orientation-selective DEER using rigid spin labels, cofactors, metals, and clusters

The dipolar interaction between two paramagnetic centres depends upon their spin–spin distance and relative orientation. Generally most experiments are carried out under conditions where the DEER signal only reports on the spin–spin distances and, for this type of data, sophisticated analysis methods for obtaining distance distributions have been developed. Recently there have been an increasing number of studies on systems where the DEER signals depend upon both distance and spin pair orientation. These investigations have relied on the use of rigid spin labels (those with a well-defined spatial position) and/or spectrometers operating at Q-band frequencies and above capable of performing DEER experiments with high resolution and sensitivity. In this article we discuss in detail orientation-selective DEER experiments for which the modulation depth and the dipolar frequencies depend on the relative orientation of the two paramagnetic centres and the distance. Analysis of the data in the presence of distance and orientation distributions is discussed, and representative examples from the literature are given for systems containing spin labels, organic cofactors, metals, and metal clusters.

Characterisation of the paramagnetic [2Fe–2S]+ centre in palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris CGA009: g-matrix determination and spin coupling analysis

Palustrisredoxin-B (PuxB) from Rhodopseudomonas palustris (CGA009) is a [2Fe-2S] ferredoxin which is able to accept electrons from NADH via the flavin-dependent palustrisredoxin reductase (PuR); these electrons can then be transferred to the P450 enzyme (CYP199A2). This work reports on the paramagnetic state of the [2Fe-2S](+) cluster in PuxB, both alone and in the PuR-PuxB complex. Aided by the X-ray crystal structure of PuxB, the protons nearest to the reduced [2Fe-2S](+) cluster were used as magnetic probes to quantify the g-matrix orientation and anisotropic magnetic moment of the paramagnetic centre. (1)H hyperfine couplings were measured with W-band Davies ENDOR and X-band HYSCORE spectroscopy and fitted to a model in which (1)H dipolar couplings were calculated assuming point magnetic moments located at the Fe ions, and bridging and coordinating cysteine sulfur atoms. The absolute sign of a (1)H hyperfine coupling was measured using a variable mixing time ENDOR experiment to confirm the assignment of the Fe(3+) and Fe(2+) ions. For the anti-ferromagnetically coupled cluster the magnetic moment is described in terms of spin projection factors, and our analysis yields values of K(exp)(A) = +2.33 to +1.85 (ferric site), and K(exp)(B) = -1.33 to -0.85 (ferrous site). These values are discussed in terms of the delocalisation of the spin density and hence the limitations of applying a local site spin coupling model to calculate the spin projection factors in a complex with considerable overlap of the α- and β-spin magnetic oribitals. The accurate description of the g-matrix orientation and magnetic moment of this [2Fe-2S](+) cluster enable it to be utilised as a paramagnetic spin probe, for example, to measure electron-electron distances. In the pdb reference frame of PuxB (code ) the g(∥) axis vector is g(∥) = [-0.6524 ± 0.0248, -0.6269 ± 0.0115, 0.4259 ± 0.0405], with the principal g-values of g(⊥) = 1.9328 ± 0.0003, g(∥) = 2.0233 ± 0.0003.

The Dynamics of Lysozyme from Bacteriophage Lambda in Solution Probed by NMR and MD Simulations

15N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and HN–Hα coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (λ lysozyme) in solution. The lower and upper lip regions in λ lysozyme (residues 51–60 and 128–141, respectively) show reduced 1H–15N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T2 indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for λ lysozyme indicate that two fluctuating β‐strands (β3 and β4) are populated in the lower lip region while the N terminus of helix α6 (residues 136–139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the β‐sheet and helical secondary structure in the lip regions, with populations of 40–60 %. Thus in solution λ lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein.

Creative visualisation in chemistry

Visualisation of molecules in the field of chemistry has been important for understanding their structure, whether simple or complicated. Initially, this was done by hand, but latterly software has come to the aid of researchers and the vast majority of chemistry visualisation is now computer-generated. As well as aiding understanding, many molecules, especially if complex in nature, can take on an artistic quality when visualised, using artificial colour for example. Often these are used for creative reasons on the front of chemistry journals, for example, and sometimes as an inspiration for more pure art forms. This paper introduces molecular graphics in the context of creative computing. It also provides a history of the development of visualisation in chemistry, especially more recently with the use of software and the increasing use on journal covers. A brief survey of some of the software involved is included. Finally, some conclusions are drawn with respect to the creative directions being taken now and possible directions in the future.

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron-Electron Resonance Spectroscopy

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe2S2] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe2S2] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.