X-BAND AND Q-BAND EPR STUDIES ON THE 2 MN-2-SUBSTITUTED METAL-BINDINGSITES 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 be en studied with Mn2+-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 si te. With the low-affinity A site unoccupied, the coordination sphere o f Mn2+ in the B site is quite distorted giving rise to a highly anisot ropic 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 param eter E/D between 0.2 and 0.3. Occupation of both binding sites with Mn 2+ induces a significant shift towards a higher symmetry in the coordi nation sphere of the B site resulting in similar zfs parameters for bo th binding sites. The change in A-site environment caused by B-site oc cupation 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 ligan d environment with small zfs parameters (D = 12 mT, E/D < 0.15). Subst ituting Co2+ with Cd2+ or Pb2+ in the B site leads to a drastic increa se in the zfs parameters of Mn2+ in the A site. The distortions are di rectly linked to the ionic radii of the ions bound to the B site and m ay be mediated by the carboxylate group of Glu216 that bridges the met al-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, th e 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.