Jonathan R. Woodward

Elucidation of the Mechanism by Which Catecholamine Stress Hormones Liberate Iron from the Innate Immune Defense Proteins Transferrin and Lactoferrin

The ability of catecholamine stress hormones and inotropes to stimulate the growth of infectious bacteria is now well established. A major element of the growth induction process has been shown to involve the catecholamines binding to the high-affinity ferric-iron-binding proteins transferrin (Tf) and lactoferrin, which then enables bacterial acquisition of normally inaccessible sequestered host iron. The nature of the mechanism(s) by which the stress hormones perturb iron binding of these key innate immune defense proteins has not been fully elucidated. The present study employed electron paramagnetic resonance spectroscopy and chemical iron-binding analyses to demonstrate that catecholamine stress hormones form direct complexes with the ferric iron within transferrin and lactoferrin. Moreover, these complexes were shown to result in the reduction of Fe(III) to Fe(II) and the loss of protein-complexed iron. The use of bacterial ferric iron uptake mutants further showed that both the Fe(II) and Fe(III) released from the Tf could be directly used as bacterial nutrient sources. We also analyzed the transferrin-catecholamine interactions in human serum and found that therapeutically relevant concentrations of stress hormones and inotropes could directly affect the iron binding of serum-transferrin so that the normally highly bacteriostatic tissue fluid became significantly more supportive of the growth of bacteria. The relevance of these catecholamine-transferrin/lactoferrin interactions to the infectious disease process is considered.

Continuous wave photolysis magnetic field effect investigations with free and protein-bound alkylcobalamins.

The activation of the Co-C bond in adenosylcobalamin-dependent enzymes generates a singlet-born Co(II)-adenosyl radical pair. Two of the salient questions regarding this process are: (1) What is the origin of the considerable homolysis rate enhancement achieved by this class of enzyme? (2) Are the reaction dynamics of the resultant radical pair sensitive to the application of external magnetic fields? Here, we present continuous wave photolysis magnetic field effect (MFE) data that reveal the ethanolamine ammonia lyase (EAL) active site to be an ideal microreactor in which to observe enhanced magnetic field sensitivity in the adenosylcobalamin radical pair. The observed field dependence is in excellent agreement with that calculated from published hyperfine couplings for the constituent radicals, and the magnitude of the MFE (<18%) is almost identical to that observed in a solvent containing 67% glycerol. Similar augmentation is not observed, however, in the equivalent experiments with EAL-bound methylcobalamin, where all field sensitivity observed in the free cofactor is washed out completely. Parallels are drawn between the latter case and the loss of field sensitivity in the EAL holoenzyme upon substrate binding (Jones et al. J. Am. Chem. Soc. 2007, 129, 15718-15727). Both are attributed to the rapid removal of the alkyl radical immediately after homolysis, such that there is inadequate radical pair recombination for the observation of field effects. Taken together, these results support the notion that rapid radical quenching, through the coupling of homolysis and hydrogen abstraction steps, and subsequent radical pair stabilization make a contribution to the observed rate acceleration of Co-C bond homolysis in adenosylcobalamin-dependent enzymes.

Time resolved studies of radical pairs

The effect of magnetic fields on chemical reactions through the RP (radical pair) mechanism is well established, but there are few examples, in the literature, of biological reactions that proceed through RP intermediates and show magnetic field-sensitivity. The present and future relevance of magnetic field effects in biological reactions is discussed.

Electron–Proton Decoupling in Excited‐State Hydrogen Atom Transfer in the Gas Phase

Hydrogen-release by photoexcitation, excited-state-hydrogen-transfer (ESHT), is one of the important photochemical processes that occur in aromatic acids and is responsible for photoprotection of biomolecules. The mechanism is described by conversion of the initial state to a charge-separated state along the O(N)-H bond elongation, leading to dissociation. Thus ESHT is not a simple H-atom transfer in which a proton and a 1s electron move together. Here we show that the electron-transfer and the proton-motion are decoupled in gas-phase ESHT. We monitor electron and proton transfer independently by picosecond time-resolved near-infrared and infrared spectroscopy for isolated phenol-(ammonia)5 , a benchmark molecular cluster. Electron transfer from phenol to ammonia occurred in less than 3 picoseconds, while the overall H-atom transfer took 15 picoseconds. The observed electron-proton decoupling will allow for a deeper understanding and control of of photochemistry in biomolecules.

Optical Absorption and Magnetic Field Effect Based Imaging of Transient Radicals

Short-lived radicals generated in the photoexcitation of flavin adenine dinucleotide (FAD) in aqueous solution at low pH are detected with high sensitivity and spatial resolution using a newly developed transient optical absorption detection (TOAD) imaging microscope. Radicals can be studied under both flash photolysis and continuous irradiation conditions, providing a means of directly probing potential biological magnetoreception within sub-cellular structures. Direct spatial imaging of magnetic field effects (MFEs) by magnetic intensity modulation (MIM) imaging is demonstrated along with transfer and inversion of the magnetic field sensitivity of the flavin semiquinone radical concentration to that of the ground state of the flavin under strongly pumped reaction cycling conditions. A low field effect (LFE) on the flavin semiquinone-adenine radical pair is resolved for the first time, with important implications for biological magnetoreception through the radical pair mechanism.

Biphotonic photochemistry of benzophenones in dimethylsulphoxide: a flash photolysis EPR study

Benzophenone and its derivatives in dimethylsulphoxide (DMSO) exhibit biphotonic photochemistry under 355 nm laser photolysis. Flash photolysis electron paramagnetic resonance experiments demonstrate that a single laser pulse is capable of producing and exciting benzophenone triplets, which can sensitize dimethylsulphoxide and subsequently lead to photodecomposition. In decafluorobenzophenone, electron transfer is the dominant process of the highly excited triplet state. Despite the rapid radiationless decay of 2-hydroxybenzophenone (2OHbenzophenone) in non-polar solvents, radical signals are observed from the photoexcitation of 2OHbenzophenone in DMSO. This is attributed to the sufficiently rapid excitation of the triplet state, which competes with the radiationless decay process, aided by the unique solvent properties of DMSO. It is concluded, in contrast to literature data, that the excited triplet state of DMSO is reactive, and can produce methyl radicals that show triplet mechanism polarization via the biphotonic photoexcitation of benzophenone.

Rapid rise time pulsed magnetic field circuit for pump-probe field effect studies

Here we describe an electronic circuit capable of producing rapidly switched dc magnetic fields of up to 20 mT with a rise time of 10 ns and a pulse length variable from 50 ns to more than 10 micros, suitable for use in the study of magnetic field effects on radical pair (RP) reactions. This corresponds to switching the field on a time scale short relative to the lifetime of typical RPs and maintaining it well beyond their lifetimes. Previous experiments have involved discharging a capacitor through a low inductance coil for a limited time using a switching circuit. These suffer from decaying field strength over the duration of the pulse given primarily by the ratio of the pulse width to the RC constant of the circuit. We describe here a simple yet elegant solution that completely eliminates this difficulty by employing a feedback loop. This allows a constant field to be maintained over the entire length of the pulse.

Alternative source of emissive CIDEP in the TREPR spectra of benzophenone in alcohols

The flash photolysis EPR spectra of benzophenone (BP) in alcoholic solvents differ in terms of their chemically induced dynamic electron polarization (CIDEP) signatures when BP is photo-excited at different laser wavelengths. The difference is strongly dependent upon the concentration of BP and is attributed to the reaction of two BP triplet states to form a BP radical anion, BP radical cation pair which occurs on a timescale sufficiently rapid relative to relaxation in the BP triplet to transfer polarization into the resulting radicals. This polarized signal dominates the spectrum obtained by 266 nm photo-excitation due to the much larger extinction coefficient of BP at this wavelength.

A two-color tunable infrared/vacuum ultraviolet spectrometer for high-resolution spectroscopy of molecules in molecular beams

We describe here the key technical elements of a two-color tunable IR/VUV photoionization TOF mass spectrometer system which allows a wide-range of high-resolution experiments to be performed on a diverse range of cold molecules and clusters in a molecular beam. In particular we highlight the methods we have applied to provide efficient wavelength separation of the VUV radiation from the longer wavelength components used to generate it and discuss a number of systems that we have studied with the instrument which highlight its flexibility for use in the study of molecular spectroscopy.

Time-resolved optical absorption microspectroscopy of magnetic field sensitive flavin photochemistry

The photochemical reactions of blue-light receptor proteins have received much attention due to their very important biological functions. In addition, there is also growing evidence that the one particular class of such proteins, the cryptochromes, may be associated with not only a biological photo-response but also a magneto-response, which may be responsible for the mechanism by which many animals can respond to the weak geomagnetic field. Therefore, there is an important scientific question over whether it is possible to directly observe such photochemical processes, and indeed the effects of weak magnetic fields thereon, taking place both in purified protein samples in vitro and in actual biochemical cells and tissues. For the former samples, the key lies in being able to make sensitive spectroscopic measurements on very small volumes of samples at potentially low protein concentrations, while the latter requires, in addition, spatially resolved measurements on length scales smaller than typical cellular components, i.e., sub-micron resolution. In this work, we discuss a two- and three-color confocal pump-probe microscopic approach to this question which satisfies these requirements and is thus useful for experimental measurements in both cases.