ORIENTATIONALLY-SELECTED 2-DIMENSIONAL ESEEM SPECTROSCOPY OF THE RIESKE-TYPE IRON-SULFUR CLUSTER IN 2,4,5-TRICHLOROPHENOXYACETATE MONOOXYGENASE FROM BURKHOLDERIA-CEPACIA AC1100

Burkholderia cepacia AC1100 is able to use the chlorinated compound 2, 4,5-trichlorophenoxyacetic acid (2,4,5-T) as the sole source of carbon and energy. CW EPR and one-dimensional ESEEM spectroscopy studies per formed earlier indicate the presence of a Rieske-type [2Fe-2S] cluster with two coordinated histidine residues in 2,4,5-T monooxygenase from B. cepacia. This paper describes the application of two-dimensional E SEEM (called HYSCORE) spectroscopy for further characterization of the nitrogens surrounding the reduced Rieske-type cluster. The HYSCORE sp ectra measured at field positions in the neighborhood of the principal directions of the g tensor contain major contributions from cross-pea ks correlating the two double-quantum transitions from each histidine nitrogen. These allow the estimation of the diagonal components of the hyperfine tensors along the principal axes of the g tensor: 4.05, 3.8 8, and 4.01 MHz (N1) and 4.71, 5.07, and 5.02 MHz (N2). Other spectral features from the histidine nitrogens usually have a much weaker inte nsity and are occasionally observed in the spectra. HYSCORE measuremen ts have been also performed with the reduced [2Fe-2S] plant ferredoxin -type cluster with four cysteine ligands in a ferredoxin from Porphira umbilicalis, and spectral features produced by the peptide nitrogen a re observed. Similar features also appear in the HYSCORE spectra of th e Rieske cluster. Systematic differences are observed between 2,4,5-T monooxygenase and published results from related benzene and phthalate dioxygenases that may reflect structural and functional differences i n histidine ligation and the nitrogens of nearby amino acids in Rieske -type [2Fe-2S] clusters.