X- and Q-band EPR studies on the two Mn(2+)-substituted metal-binding sites of D-xylose isomerase.

The two metal-binding sites (A and B)/subunit of the homotetrameric D-xylose isomerase (Xyl isomerase) from Streptomyces rubiginosus have been studied with Mn(2+)-EPR spectroscopy at X-band and Q-band frequencies and with electronic spectroscopy. Displacement studies in the visible absorbance range showed that Mn2+ have a higher affinity for the B site. With the low-affinity A site unoccupied, the coordination sphere of Mn2+ in the B site is quite distorted giving rise to a highly anisotropic X-band EPR spectrum. Simulation of the Q-band spectrum reveals a zero field splitting (zfs) D of about 45-48 mT and a rhombicity parameter E/D between 0.2 and 0.3. Occupation of both binding sites with Mn2+ induces a significant shift towards a higher symmetry in the coordination sphere of the B site resulting in similar zfs parameters for both binding sites. The change in A-site environment caused by B-site occupation was analysed in mixed Xyl isomerase derivatives, in which the B site is loaded with Co2+, Cd2+ or Pb2+ and the A site with Mn2+. In the Co2+/Mn2+ Xyl isomerase the Mn2+ has a relatively symmetric ligand environment with small zfs parameters (D = 12 mT, E/D < 0.15). Substituting Co2+ with Cd2+ or Pb2+ in the B site leads to a drastic increase in the zfs parameters of Mn2+ in the A site. The distortions are directly linked to the ionic radii of the ions bound to the B site and may be mediated by the carboxylate group of Glu216 that bridges the metal-binding sites. The EPR spectra also reflect the catalytic activity of the mixed metal samples. With the larger Cd2+ or Pb2+ in the B site, which are strongly influencing the stereochemistry of the A site, the catalytic activity is lost, whereas Co2+ and Mn2+ render the enzyme in an active state, so that the mutual influence on catalysis depends on the complex geometry of both metal-binding sites.

Visible, EPR and electron nuclear double-resonance spectroscopic studies on the two metal-binding sites of oxovanadium (IV)-substituted D-xylose isomerase.

The two metal-binding sites of the D-xylose isomerase from Streptomyces rubiginosus were studied using VO2+ as a sensor for the ligand environment. Titration of the tetrameric enzyme with VO2+, followed by EPR spectroscopy and inhibition studies, show that the first four VO2+ equivalents occupy, in analogy to Co2+, Cd2+ and Pb2+, the binding site B. The visible absorption data and the EPR parameters indicate that a nitrogen ligand is involved in the ligand sphere of the high-affinity B site. The low-affinity A site could be studied selectively by blocking the B site with visible and EPR-silent Cd2+. The visible data and EPR parameters for this site are consistent with a ligand environment composed of oxygen donors without nitrogen ligation. The nitrogen coordination in the high-affinity site could be demonstrated by electron nuclear double-resonance (ENDOR) studies of the 4VO2+ enzyme, and was assigned to a histidine ligand. The 14N resonances are interpreted in terms of a quartet with a coupling value of 13.2 MHz. 1H-ENDOR coupling of 1.7 MHz, exchangeable in D2O, has been assigned to the N-H proton of the histidine. Additional proton ENDOR couplings, which are not exchangeable, are due to protons bound to the carbon atoms of the histidine. For the low-affinity binding site, a nitrogen coordination could be definitely excluded by the ENDOR measurements. Exchangeable 1H-ENDOR couplings observed in this sample were assigned to H2O ligands in the vicinity of VO2+. The results closely relate to what is known from X-ray structure. However, the relative affinities for the two binding sites seem not to be the same for different bivalent cations. In mixed metal samples with four VO2+ and four Co2+ equivalents, the VO2+ is distributed between both binding sites. Small changes in the complex geometry of the A site, indicated by different EPR features, seem to occur if the B site is occupied by Co2+ or by Cd2+.

Spectroscopic studies on the metal-ion-binding sites of Co2(+)-substituted D-xylose isomerase from Streptomyces rubiginosus.

The coordination sphere of the two metal-binding sites/subunit of the homotetrameric D-xylose isomerase from Streptomyces rubiginosus has been probed by the investigation of the Co2(+)-substituted enzyme using electronic absorption, CD and magnetic circular dichroic spectroscopies in the visible region. The spectrum of the high-affinity site (B site) has an absorption coefficient, epsilon 545, of 18 M-1 cm-1, indicating a distorted octahedral complex geometry. The spectrum of the low-affinity site (A site) shows two absorption maxima at 505 nm and 586 nm with epsilon values of 170 M-1 cm-1 and 240 M-1 cm-1, respectively, which indicates a distorted tetrahedral or pentacoordinated complex structure as also observed for the enzyme from Streptomyces violaceoruber [Callens et al. (1988) Biochem. J. 250, 285-290] having the same feature but lower epsilon values. The first 4 mol Co2+ added/mol apoenzyme occupy both sites nearly equally. Subsequently the Co2+ located in the A site slowly moves into the B site. After equilibrium is reached, the next 4 mol Co2+/mol again occupy the A site with its typical spectrum, restoring full activity. Addition of 4 mol Cd2+ or Pb2+/mol Co4-loaded derivative displaces the Co2+ from the B site to form the Pb4/Co4 derivative containing Co2+ in the A site, reducing activity fourfold while the Pb4/Pb4 species is completely inactive. In contrast, Eu3+ displaces Co2+ preferentially from the A site. Thus, the high- and low-affinity sites may be different for different cations. After addition of the substrates D-xylose, D-glucose and D-fructose and the inhibitor xylitol the intense Co2+ A-site spectrum of both the active Co4/Co4 derivative and the less active Pb4/PCo4 derivative decreases, indicating that these compounds are bound to the A site, changing the distorted tetrahedral or pentacoordinated symmetry there to a distorted octahedral complex geometry.