Jeffrey Harmer

How Escherichia coli Is Equipped to Oxidize Hydrogen under Different Redox Conditions

The enterobacterium Escherichia coli synthesizes two H2 uptake enzymes, Hyd-1 and Hyd-2. We show using precise electrochemical kinetic measurements that the properties of Hyd-1 and Hyd-2 contrast strikingly, and may be individually optimized to function under distinct environmental conditions. Hyd-2 is well suited for fast and efficient catalysis in more reducing environments, to the extent that in vitro it behaves as a bidirectional hydrogenase. In contrast, Hyd-1 is active for H2 oxidation under more oxidizing conditions and cannot function in reverse. Importantly, Hyd-1 is O2 tolerant and can oxidize H2 in the presence of air, whereas Hyd-2 is ineffective for H2 oxidation under aerobic conditions. The results have direct relevance for physiological roles of Hyd-1 and Hyd-2, which are expressed in different phases of growth. The properties that we report suggest distinct technological applications of these contrasting enzymes.

Size dependent oxygen buffering capacity of ceria nanocrystals

The structural, chemical and electronic changes of ceria as a function of decreasing particle size have been studied: at sizes below 5 nm the total amount of reducible oxygen dramatically increases due to superoxide formation on the ceria surface, a result indicative of a size dependent oxygen buffering capacity.

Sodium phosphaethynolate, Na(OCP), as a “P” transfer reagent for the synthesis of N-heterocyclic carbene supported P3 and PAsP radicals

Sodium phosphaethynolate, Na(OCP), reacts as a P− transfer reagent with the imidazolium salt [DippNHC–H][Cl] [DippNHC = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene] to give the parent phosphinidene–carbene adduct, DippNHCPH, with the loss of CO. In a less atom economic reaction, the cage compound, P7(TMS)3 (TMS = SiMe3) reacts likewise with the imidazolium salt to yield DippNHCPH thereby giving two entry points into parent phosphinidene-based chemistry. From the building block DippNHCPH, the carbene-supported P3 cation [(DippNHC)2(μ-P3)][Cl] was rationally synthesized using half an equivalent of PCl3 in the presence of DABCO (1,4-diazabicyclo[2.2.2]octane). The corresponding arsenic analogue, [(DippNHC)2(μ-PAsP)][Cl], was synthesized in the same manner using AsCl3. The reduction of both [(DippNHC)2(μ-P3)][Cl] and [(DippNHC)2(μ-PAsP)][Cl] into their corresponding neutral radical species was achieved simply by reducing the compounds with an excess of magnesium. This allowed the electronic structures of the compounds to be investigated using a combination of NMR and EPR spectroscopy, X-ray crystallography, and computational studies. The findings of the investigation into (DippNHC)2(μ-P3) and (DippNHC)2(μ-PAsP) reveal the central pnictogen atom in both cases as the main carrier of the spin density (∼60%), and that they are best described as the P3 or PAsP analogues of the elusive allyl radical dianion. The phosphorus radical was also able to undergo a cycloaddition with an activated acetylene, followed by an electron transfer to give the ion pair [(DippNHC)2(μ-P3)][P3(C(COOMe))2].

Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron–electron resonance

In oxidative phosphorylation, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. Complex I contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters, in several subunits, to transfer the electrons to quinone. Understanding coupled electron transfer in complex I requires a detailed knowledge of the properties of individual clusters and of the cluster ensemble, and so it requires the correlation of spectroscopic and structural data: This has proved a challenging task. EPR studies on complex I from Bos taurus have established that EPR signals N1b, N2 and N3 arise, respectively, from the 2Fe cluster in the 75 kDa subunit, and from 4Fe clusters in the PSST and 51 kDa subunits (positions 2, 7, and 1 along the seven-cluster chain extending from the flavin). The other clusters have either evaded detection or definitive signal assignments have not been established. Here, we combine double electron-electron resonance (DEER) spectroscopy on B. taurus complex I with the structure of the hydrophilic domain of Thermus thermophilus complex I. By considering the magnetic moments of the clusters and the orientation selectivity of the DEER experiment explicitly, signal N4 is assigned to the first 4Fe cluster in the TYKY subunit (position 5), and N5 to the all-cysteine ligated 4Fe cluster in the 75 kDa subunit (position 3). The implications of our assignment for the mechanisms of electron transfer and energy transduction by complex I are discussed.

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.

Structural model for the protein-translocating element of the twin-arginine transport system

Significance The twin-arginine transport system (Tat) has the remarkable ability of transporting folded proteins across membranes while avoiding uncontrolled ion leakage. Tat is essential for plant photosynthesis and is required for bacterial pathogenesis. The mechanism by which folded proteins are translocated is poorly understood. We have determined the structure of the TatA oligomer, which is responsible for the translocation step, and evaluated its impact on lipid bilayers. The results suggest a mechanism of protein translocation involving thinning and perturbing the membrane bilayer. The approach used here will be useful for structural analysis of other oligomeric proteins that weakly assemble in the membrane. The twin-arginine translocase (Tat) carries out the remarkable process of translocating fully folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Tat is required for bacterial pathogenesis and for photosynthesis in plants. TatA, the protein-translocating element of the Tat system, is a small transmembrane protein that assembles into ring-like oligomers of variable size. We have determined a structural model of the Escherichia coli TatA complex in detergent solution by NMR. TatA assembly is mediated entirely by the transmembrane helix. The amphipathic helix extends outwards from the ring of transmembrane helices, permitting assembly of complexes with variable subunit numbers. Transmembrane residue Gln8 points inward, resulting in a short hydrophobic pore in the center of the complex. Simulations of the TatA complex in lipid bilayers indicate that the short transmembrane domain distorts the membrane. This finding suggests that TatA facilitates protein transport by sensitizing the membrane to transient rupture.

Stable GaX2, InX2 and TlX2 radicals

The chemistry of the Group 13 metals is dominated by the +1 and +3 oxidation states, and simple monomeric M(II) species are typically short-lived, highly reactive species. Here we report the first thermally robust monomeric MX2 radicals of gallium, indium and thallium. By making use of sterically demanding boryl substituents, compounds of the type M(II)(boryl)2 (M = Ga, In, Tl) can be synthesized. These decompose above 130 °C and are amenable to structural characterization in the solid state by X-ray crystallography. Electron paramagnetic resonance and computational studies reveal a dominant metal-centred character for all three radicals (>70% spin density at the metal). M(II) species have been invoked as key short-lived intermediates in well-known electron-transfer processes; consistently, the chemical behaviour of these novel isolated species reveals facile one-electron shuttling processes at the metal centre.

Development of a Metal‐Ion‐Mediated Base Pair for Electron Transfer in DNA

A new C-nucleoside structurally based on the hydroxyquinoline ligand was synthesized that is able to form stable pairs in DNA in both the absence and the presence of metal ions. The interactions between the metal centers in adjacent Cu(II)-mediated base pairs in DNA were probed by electron paramagnetic resonance (EPR) spectroscopy. The metal-metal distance falls into the range of previously reported values. Fluorescence studies with a donor-DNA-acceptor system indicate that photoinduced charge-transfer processes across these metal-ion-mediated base pairs in DNA occur more efficiently than over natural base pairs.

High-resolution EPR spectroscopic investigations of a homologous set of d9-cobalt(0), d9-rhodium(0), and d9-iridium(0) complexes.

The 17-electron complexes [M(tropp(ph))2] (M=Co0, Rh0, Ir0) were prepared and isolated (tropp = tropylidene phosphane). A structural analysis of [Co(tropp(ph))2] revealed this complex to be almost tetrahedral, while the heavier homologues have more planar structures. Partially deuterated tropp complexes [D6][M(tropp(ph))2] were synthesised for M = Rh and Ir in order to enhance the resolution in the EPR spectra. This synthesis involves a four-fold intramolecular C-H activation reaction, whereby alkyl groups are transformed into olefins. Dihydrides were observed as intermediates for M = Ir. The electronic and geometric structures of all complexes [M(tropp(ph))2] (M = Co, Rh, Ir) and [D6][M(tropp(ph))2] (M = Rh, Ir) were investigated by continuous wave (CW) and echo-detected EPR in combination with pulse ENDOR and ESEEM techniques. In accord with their planar structures, cis and trans isomers were detected for [M(tropp(ph))2] (M = Rh0, Ir0) for which a dynamic equilibrium was established. The thermodynamic data show that the cis isomer is slightly preferred by deltaH(o) = -4.7 +/- 0.3 kJ mol(-1) (M = Rh) and delta H(o) = -5.1 +/- 0.5 kJ mol(-1); (M = Ir). The entropies for the process trans-[M(tropp(ph))2] <==> cis-[M(tropp(ph))2] are also negative [deltaS(o) = -5 +/- 1.5 J mol(-1) (M = Rh); deltaS(o) = -17 +/- 3.7 J mol(-1) (M = Rh)], indicating higher steric congestion in the cis isomers. The cobalt(0) and irdium(0) complexes show rather large g anisotropies, while that of the rhodium(0) complex is small (Co: g(parallel) = 2.320, g(perpendicular) = 2.080; cis-Rh: g(parallel) = 2.030, g(perpendicular) = 2.0135; trans-Rh: g(parallel) = 2.050, g(perpendicular) = 2.030; cis-Ir: g(parallel) = 2.030, g(perpendicular) = 2.060; trans-Ir: g(parallel) = 1.980, g(perpendicular) = 2.150). The g matrices of [M(tropp(ph))2] (M = Co, Rh) are axially symmetric with g(parallel) > g(perpendicular), indicating either a distorted square planar structure (SOMO essentially d(x2 - y2) or a compressed tetrahedron (SOMO essentially d(xy)). Interestingly, for [Ir(tropp(ph))2] the inverse ordering, g(perpendicular) > g(parallel) is found; this cannot be explained by simple ligand field arguments and must await a more sophisticated analysis. The hyperfine interactions of the unpaired electron with the metal nuclei, phosphorus nuclei, protons, deuterons and carbon nuclei were determined. By comparison with atomic constants, the spin densities on these centres were estimated and found to be small. However, the good agreement of the distance between the olefinic protons and the metal centres determined from the dipolar coupling parameter indicates that the unpaired electron is primarily located at the metal centre.

Binding of Coenzyme B Induces a Major Conformational Change in the Active Site of Methyl-Coenzyme M Reductase

Methyl-coenzyme M reductase (MCR) is the key enzyme in methane formation by methanogenic Archaea. It converts the thioether methyl-coenzyme M and the thiol coenzyme B into methane and the heterodisulfide of coenzyme M and coenzyme B. The catalytic mechanism of MCR and the role of its prosthetic group, the nickel hydrocorphin coenzyme F(430), is still disputed, and no intermediates have been observed so far by fast spectroscopic techniques when the enzyme was incubated with the natural substrates. In the presence of the competitive inhibitor coenzyme M instead of methyl-coenzyme M, addition of coenzyme B to the active Ni(I) state MCR(red1) induces two new species called MCR(red2a) and MCR(red2r) which have been characterized by pulse EPR spectroscopy. Here we show that the two MCR(red2) signals can also be induced by the S-methyl- and the S-trifluoromethyl analogs of coenzyme B. (19)F-ENDOR data for MCR(red2a) and MCR(red2r) induced by S-CF(3)-coenzyme B show that, upon binding of the coenzyme B analog, the end of the 7-thioheptanoyl chain of coenzyme B moves closer to the nickel center of F(430) by more than 2 A as compared to its position in both, the Ni(I) MCR(red1) form and the X-ray structure of the inactive Ni(II) MCR(ox1-silent) form. The finding that the protein is able to undergo a conformational change upon binding of the second substrate helps to explain the dramatic change in the coordination environment induced in the transition from MCR(red1) to MCR(red2) forms and opens the possibility that nickel coordination geometries other than square planar, tetragonal pyramidal, or elongated octahedral might occur in intermediates of the catalytic cycle.