ENDOR evidence relating the Fe/S EPR signals from xanthine oxidase to the individual clusters.

Our knowledge of xanthine oxidase, the most widely studied enzyme containing the pterin molytxlenum cofactor [l]. has recently been greatly enhanced by the X-ray crystallographic determination of the structure of the closely related aldehyde oxidoreductase from Desulfov/btfo gbas [2,3]. As a result of EPR work, it has long been recognised that these enzymes contain two kinds of pFe2SI centres, called F& I and FeB II, whlch are EPR active in their reduced states. Fels I has EPR parameters similar to those of spinach ferredoxln [4], with gw of 1.95, whereas Fe/S II is unusual In having a g, of 2.00 and not being observable at temperatures above about 40 K. The crystal structure also reveals two kinds of [2Fe-2SI centres, one with a similar fold to that in spinach ferredoxin, and the other having a (so far) unique fold; the former is on the surface of the molecule and exposed to the solvent, whereas the latter is completely buried. It is not known which Fe-S centre seen in the X-ray structure corresponds to which spectrascopically distinct Fels centre. To determine this, we have undertaken a study of the exchangeability of protons interacting with these Fe/S centres by using ENDOR (Electron Nudear Double Resonance), a form of spectroscopy in which coupling between magnetlc nudei and unpaired electrons is observed [5]. ENDOR spectra were measured and xanthine oxidase prepared as described previously [61. The enzyme was reduced for 15 minutes by 4 mM sodium dithlonite in 50 mM, pHQD 8.2 Na bldne buffer containing 1 mM EDTA. Exchange into ' H,O buffer was done using a G25 column. The sharp features in the EPR spectrum on the right of Fig 1 are due to Fe/S 1. The Fe/S II spectrum largely underlies that of Fe/S