Introduction: The BphC enzyme (2,3-dihydroxybiphenyl dioxygenase) is a key enzyme in the PCB (polychlorinated biphenyls) degradation pathway of microorganisms. This enzyme is one of the extradiol type catechol ring cleavage dioxygenases having non-heme iron (Fe(II)) site in each of the eight subunits. It cleaves the catechol ring of its substrate, 2,3-dihydroxybiphenyl (2,3-DHBP). To gain insights into the reaction mechanism of these enzymes, we undertook X-ray crystallographic analysis of the BphC enzyme from Pseudomonas sp. strain KKS102. Three-dimensional structure was determined at 1.8 9 resolution. Furthermore, we have prepared more than 20 mutant proteins. The results of our biochemical and crystallographic analyses of the native and mutant BphC enzymes follow.Active site and substrate binding: The Fe ion coordinates the side-chains of the following amino acid residues, His145, His209 and Glu260, as well as two water molecules with a square-pyramidal coordination geometry. Upon binding the substrate, the two water molecules are replaced with the hydroxyl groups of the substrate, 2,3-DHBP. The resultant coordination polyhedra may be described as a distorted trigonal bipyramid.Mutations on the amino acid residues bound to Fe ion resulted in the lack of Fe ion, which, however, did not spoil the ability for binding substrates, suggesting that protein surface complementarity for the substrates suffices for binding.The substrate binding pocket is highly complementary to the substrate molecule, 2,3-DHBP. Substitutions changing the shape and/or size of the pocket were found to reduce the enzymatic activity. Comparing the crystal structures of various mutant BphC enzymes together with the corresponding enzyme kinetics data, we feel that the complementarity between the enzyme and substrate surfaces strongly affect the catalytic activities exhibited by the mutant enzymes.Oxygen binding site: For the enzymatic reaction to occur, the participation of an O2 molecule is required. Based on the present crystal structures, we proposed a three-dimensional model for the ternary complex composed of the BphC enzyme, a substrate and an O2 molecule. Considering the van der Waals radii of the atoms around the Fe ion, and proposed reaction mechanism based on spectroscopic studies, the cavity around Ala197 and Val147 appears to be the O2 binding site, which would be conveniently located for an O2 molecule to attack 2,3-DHBP.His194 is a critical residue for catalysis: From mutational analyses of amino acid residues around the catechol ring moiety of the substrate, His194 has been identified as a critical residue for catalysis. Most notably H194F mutant shows no enzymatic activity. The crystal structure of this mutant shows that His194 is located adjacent to the hydroxyl group of the substrate, and little conformation change occurs around the active site upon the H194F mutation. Thus, most probably, His194 is the proton acceptor as suggested from previous spectroscopic studies.