Pyruvate oxidase (POX) from L. plantarum contains the cofactor flavin-adeninedinucleotide (FAD). The isoalloxazine ring of FAD is normally planar, yet is found in a bent conformation in POX. The effect of this bend on the properties of FAD was investigated by redox-induced Fourier-Transform-Infrared difference spectroscopy, using the free cofactor and several flavoproteins and POX variants. Some bands of the redox-induced IR difference spectra could be assigned to vibrational modes, based on a normal mode analysis of several lumiflavin species, using a density functional theoretical approach. Frequency differences of some modes between free and enzymebound FAD are discussed. Enzyme kinetical and IR-spectroscopic properties of the POX variant V265A are explained on the basis of the crystal structure which has been determined in the present work. The second cofactor of POX is thiamin diphosphate (ThDP). The catalytic function of several amino acid residues in the vicinity of ThDP was determined by measuring steady state concentrations of ThDP reaction intermediates for a number of POX variants. Furthermore, the specificity of POX regarding the substrate phosphate and the coupling of the electron transfer step with the transfer of the acetyl moiety from acetyl-ThDP to phosphate or other nucleophiles were investigated.