ESR/EPR can provide unique information on the identity and structure of stable and transient paramagnetic species such as defects, metals, clusters and organic cofactors, and nitroxide spin-labels engineered into proteins by, for example, ENDOR (electron-nuclear double resonance) and HYSCORE (hyperfine correlation spectroscopy). In addition, pulsed ELDOR (electron-electron double resonance) can measure distances between electron spins over the range 15 – 80 Å. Furthermore, the time-domain on the nanosecond scale can be accessed by initiation of reactions using a laser pulse that simultaneously triggers detection by pulsed EPR.
Recent publications
- "Structure of the Pyrroloquinoline Quinone Radical in Quinoprotein Ethanol Dehydrogenase" [PDF File] C. W. M. Kay, B. Mennenga, H. Görisch, and R. Bittl. Journal of Biological Chemistry 281 (2006) 1470-1476.
- "Substrate binding in quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa studied by electron-nuclear double resonance" [PDF File] C. W. M. Kay, B. Mennenga, H. Görisch and R. Bittl. Proc Natl Acad Sci (USA) 2006 (103) 5267-5272.
A number of Gram-negative bacteria utilize a class of dehydrogenases known as quinoproteins, which are distinct from the flavin- and nicotinamide-dependent enzymes, to catalyze the oxidation of alcohols or aldoses. The reaction is the first step in an electron transport chain that generates a proton motive force that is used to produce ATP. Several quinoproteins contain the noncovalently bound quinoid cofactor pyrroloquinoline quinone (PQQ), the role of which as a potential vitamin in mammals is currently under debate. The first of these papers describes the characterisation of the PQQ radical in Quinoprotein Ethanol Dehydrogenase, by advanced EPR & density functional theory. Although the structure of these proteins has been elucidated by X-ray crystallography until now the location of the alcohol substate could not be determined. In the second of these papers we established the position of the substrate in the binding pocket using the same experimental and theoretical methods.
Serum transferrin and lactoferrin are members of an important group of iron-binding and transport proteins.A single polypeptide folds into two lobes of similar structure, each binding a single Fe3+ ion. The iron can be removed and replaced by a number of other metal ions, while retaining the overall protein structure. Of great interest is how these proteins interact with their bacterial and mammalian receptors and how changes to the tertiary structure upon binding ultimately lead to iron release. In this paper, we measured the distance between the metal centers in copper-containing transferrin and lactoferrin by pulsed ELDOR. This work lays the basis for using EPR to directly observe changes in conformation in these proteins, and in other metalloproteins using the intrinsic metal centers as spin-labels, rather than having to artificially label them.
- "Blue light perception in plants. Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin" [PDF File] C. W. M. Kay, E. Schleicher, A. Kuppig, H. Hofner, W. Rüdiger, M. Schleicher, M. Fischer, A. Bacher, S. Weber, G. Richter J Biol Chem 278 (2003) 10973-10982
Numerous phenomena in the life cycle of plants such as circadian timing, regulation of gene expression, and phototropism (theadaptive process whereby plants bend toward a light source tomaximize light capture for photosynthesis) are responses to ambientlight levels in the UV-A and blue spectral regions comprisingwavelengths of about 320-500 nm. Up to the present, two classesof blue light photoreceptors have been identified in plants; theyare the cryptochromes and the phototropins,both of which are flavoproteins. In this paper we investigated the photoactivity of phototropins by advanced EPR spectroscopic methods and based on the results could suggest a radical pair mechanism as the basis for the formation of the intermediate signaling state, a covalent adduct between the flavin cofactor and nearby cysteine residue.
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