A Rieske non-heme iron ring-hydroxylating oxygenase (RHO) from Sphingobium sp. PNB involved in the initial oxidation of a wide range of low and high molecular weight polycyclic aromatic hydrocarbons (PAHs) was investigated. The RHO was shown to comprise of the gene products of distantly located ahdAlf-ahdA2f, ahdA3 and ahdA4 genes, which encoded the oxygenase alpha-and beta-subunits, ferredoxin and reductase, respectively. In silico structural analysis of AhdAlf revealed a very large substrate-binding pocket, satisfying the spatial requirements to accommodate high molecular weight substrates. In addition, an atypical substrate access channel was noticed from the topology analysis of the oxygenase. Guided by molecular docking studies, dioxygenation of several PAHs as well as alkyl-and aryl benzenes was examined with the recombinant AhdAlfA2f expressed in Escherichia coli. Chromatographic and mass spectrometric analyses confirmed that AhdAlfA2f displays broad substrate specificity towards a wide range of aromatic hydrocarbons including potential xenobiotics, demonstrating metabolic robustness of strain PNB.