MAGNETIC RESONANCE IN CHEMISTRY Magn. Reson. Chem. 2005; 43: S65–S73 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/mrc.1691 Multi-frequency high-field EPR studies on metal-substituted xylose isomerase † R. Kappl, 1 K. Ranguelova, 1 B. Koch, 1 C. Duboc 2 and J. H ¨ uttermann 1 * 1 Institut fuer Biophysik, FR 2.5, Geb. 76, Klinikum, Universit ¨ at des Saarlandes, 66421 Homburg, Germany 2 Grenoble High Magnetic Field Laboratory GHMFL, 25, Avenue des Martyrs – B.P. 166-38042 Grenoble Cedex 9, France Received 15 April 2005; Revised 29 June 2005; Accepted 30 June 2005 The bacterial enzyme D-xylose isomerase (XI) catalyses the conversion of D-xylose to D-xylulose. Each subunit of the homotetrameric protein contains a bimetallic active centre requiring divalent metal ions such as Mg 2+ , Mn 2+ or Co 2+ for catalytic activity. We report here on XI in which the metal binding site 1 is specifically loaded with EPR active Mn 2+ , while binding site 2 is occupied by Co 2+ or Cd 2+ , rendering a catalytically active or inactive species respectively. The Q-band (34 GHz) EPR spectra of these mixed-metal samples (Co 2+ /Mn 2+ and Cd 2+ /Mn 2+ XI) show a clear influence of the metal in site 2 on the Mn 2+ EPR parameters. Likewise, a systematic increase of the zero field splitting parameters (zfs) of Mn 2+ in site 1 upon incubation with the inhibitor xylitol or substrates for both mixed-metal samples is found. For Co 2+ /Mn 2+ XI complexed with substrate, a drastic line broadening of the central -1/2 $ +1/2 transition is observed in Q-band EPR, which was not amenable to analysis so far. By means of a multi-frequency approach at frequencies beyond Q-band, the relevant zfs parameters were derived from spectral simulations of EPR spectra measured at 94, 285 and 670 GHz. It is shown that parallel to the increase of the D-value its distribution also grows considerably in going from free Co 2+ /Mn 2+ XI to the species complexed with inhibitor or substrate. For XI with bound substrate, D-values in the range of 70–90 mT and a distribution of about 30 mT were found from simulation trials. The large distribution in zfs values is thought to be correlated to the structural disorder induced by the shift of the metal ion of site 2 into a location necessary for the isomerisation reaction. The results are discussed with respect to high-resolution crystal data. Copyright  2005 John Wiley & Sons, Ltd. KEYWORDS: electron paramagnetic resonance; high-field EPR; 55 Mn; xylose isomerase; zero field splitting INTRODUCTION The bacterial enzyme D-xylose isomerase (XI) catalyses the conversion of the aldose D-xylose to the ketose D-xylulose as part of the xylose metabolic pathway in micro-organisms. It also converts D-glucose to D-fructose, a reaction indus- trially applied to produce high-fructose corn syrup. 1,2 The homotetrameric enzyme contains a bimetallic centre as active site and has an absolute requirement for bivalent metal ions such as Mg 2C , Mn 2C , Fe 2C or Co 2C . 3–6 Under most conditions, Mg 2C appears to be the physiological cofac- tor, particularly for Actinomycetaceae strains, but in other bacterial species (e.g. Escherichia or Lactobacillus), XI is best activated by Mn 2C . 7–9 Extensive X-ray work on XI of different bacterial species such as Streptomyces rubiginosus, 10 – 14 Strepto- myces olivochromogenes, 15,16 Arthrobacter 17,18 and Actinoplanes missouriensis 19,20 has revealed a high structural similarity. Each subunit comprises an eight-stranded parallel ˇ-sheet † Presented as part of a special issue on High-field EPR in Biology, Chemistry and Physics. L Correspondence to: J. H ¨ uttermann, Institut f ¨ ur Biophysik, FR 2.5 Geb. 76, Universit¨ atsklinikum 66421 Homburg, Germany. E-mail: bpjhue@uniklinik-saarland.de Contract/grant sponsor: DFG priority program; Contract/grant number: SPP 1051. surrounded by eight ˛-helices (called triose phosphate iso- merase barrel motif) and an extended C-terminal helical loop that embraces the neighbouring subunit. The bimetallic active site is located at the centre of the barrel, and all residues ligating the metal ions as well as some important residues in the vicinity are conserved in all species. In the substrate-free form, a bridging glutamate residue separates the metal sites 1 and 2 by about 4.9 ˚ A. For Mn 2C -substituted XI, the liga- tion sphere of site 1 is made up by four carboxylate groups and two water molecules, whereas site 2 has a histidine, three carboxylate groups and one water molecule as ligands (Scheme 1). In several crystal structures with substrates, sub- strate analogues or inhibitors, observations relevant to the enzyme mechanism have been made. Substrates were found to be bound to the metal in site 1 replacing two water molecules, both in the extended chain 11,14,18 and in the cyclic form. 14,21 In the presence of the substrate, the metal ion of site 2 adopts more than one position, partly with low occupancies, indicating a metal movement during substrate binding and catalysis. On the basis of this structural data and in accordance with most biochemical and kinetic studies, a stepwise mechanism for the isomerisation reaction was pro- posed: (i) binding of the cyclic ˛ anomer of the substrate, (ii) enzyme-catalysed ring opening and rearrangement during Copyright  2005 John Wiley & Sons, Ltd.