COMPARATIVE EPR AND REDOX STUDIES OF 3 PROKARYOTIC ENZYMES OF THE XANTHINE-OXIDASE FAMILY - QUINOLINE 2-OXIDOREDUCTASE, QUINALDINE 4-OXIDASE, AND ISOQUINOLINE 1-OXIDOREDUCTASE

For three prokaryotic enzymes of the xanthine oxidase family, namely q uinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-ox idoreductase, the electron transfer centers were investigated by elect ron paramagnetic resonance. The enzymes are containing a molybdenum-mo lybdopterin cytosine dinucleotide cofactor, two distinct [2Fe-2S] clus ters and, apart from isoquinoline 1-oxidoreductase, a flavin adenine d inucleotide. The latter cofactor yields two different organic radical signals in quinoline 2-oxidoreductase and quinaldine 4-oxidase, typica l for the neutral and anionic form, respectively. A ''rapid'' Mo(V) sp ecies is present in all enzymes with small differences in magnetic par ameters. From spectra simulation of Mo-95-substituted quinoline 2-oxid oreductase, a deviation of 25 degrees between the maximal g and Mo-95- hyperfine tensor component was derived. The very rapid Mo(V) species w as detected in small amounts upon reduction with substrates in quinoli ne 2-oxidoreductase and quinaldine 4-oxidase, but showed a different k inetic behavior with considerable EPR intensities in isoquinoline 1-ox idoreductase. The FeSI and FeSII centers produced different signals in all three enzymes and, in case of isoquinoline 1-oxidoreductase, reve aled a dipolar interaction, from which a maximum distance of 15 Angstr om between FeSI and FeSII: was estimated. The midpoint potentials of t he FeS centers were surprisingly different and determined for FeSI/FeS II with -155/-195 mV in quinoline 2-oxidoreductase, -250/-70 mV in qui naldine 4-oxidase, and +65/+10 mV in isoquinoline 1-oxidoreductase. Th e slopes of the fitting curves for the Nernst equation are indicative for nonideal behavior. Only in quinoline 2-oxidoreductase, an averaged midpoint potential of the molybdenum redox pairs of about -390 mV cou ld be determined. Both of the other enzymes did not produce Mo(V) sign als in redox titration experiments, probably because of direct reducti on of Mo(VI) to Mo(IV) in the presence of dithionite.