Biochemical, Mossbauer, and EPR studies of the Diiron cluster of phenol hydroxylase from Pseudomonas sp strain CF 600

Phenol hydroxylase of Pseudomonas sp. strain CF600 comprises three components: DmpP is an FAD- and [2Fe-2S]-containing reductase; DmpM is a cofactorless activator protein; and DmpLNO is the oxygenase. Single turnover experiments established that DmpLNO contains the active site, but requires DmpM for efficient turnover: the steady-state turnover rate reaches a maximum at 1.5 DmpM: I DmpLNO. Chemical cross-linking experiments showed that DmpM interacts with the large subunit of the DmpLNO oxygenase complex. Mossbauer studies revealed that the active site of the oxygenase can accommodate two types of diiron clusters, each of these cluster types having two equivalent sites. Cluster form 1, representing typically around 85% of total Fe, has DeltaE(Q) = 1.73 mm/s and delta = 0.54 mm/s, while cluster II exhibits DeltaE(Q) = 0.79 mm/s and Delta = 0.48 mm/s. Studies in strong applied magnetic fields suggest that the two iron sites of cluster I are bridged by an oxo group while sites in cluster II appear to be hydroxo-bridged. Reduction of the samples with dithionite yields the diferrous forms of the clusters. Air oxidation of the reduced samples leads to an increase of the cluster II fraction, accompanied by a corresponding decrease in catalytic activity. The reduced oxygenase samples exhibit at X-band an integer spin EPR signal centered, in parallel mode, at g = 16.6. Quantitative analysis showed that 19% of the clusters contribute to the EPR signal, suggesting that cluster II is the EPR-active species. Incubation with dithiothreitol (DTT) inactivated the oxygenase by a mechanism apparently involving H2O2 generation. In addition, Mossbauer studies of DTT-inactivated enzyme showed that all ferric iron belonged to one diamagnetic diferric cluster with parameters that indicate that DTT coordinates to the cluster.