Kinetic and CD/MCD Spectroscopic Studies of the Atypical, 3-His Ligated, Non-heme Fe2+ Center in Diketone Dioxygenase: the Role of Hydrophilic Outer Shell Residues in Catalysis

Diketone cleaving enzyme (Dke1) is a dioxygenase with an atypical, 3 histidine ligated, mononuclear non-heme Fe 2+ center. To assess the role in enzyme catalysis of the hydrophilic residues in the active site pocket, residues Glu98, Arg80, Tyr70 and Thr107 were subjected to mutational analysis. Steady state and pre-steady state kinetics indicated a role for Glu98 in promoting both substrate binding and O 2 reduction. Additionally, the Glu98-substitution eliminated the pH dependence of substrate binding ( k cat app / K M app -pH profile) present in wild type Dke1 (p K a 6.3±0.4 and 8.4±0.4). MCD spectroscopy revealed that the Glu98→Gln mutation leads to the conversion of the six-coordinate (6C) resting Fe 2+ center present in the wild type enzyme at pH 7.0 to a mixture of 5C and 6C sites. The 6C geometry was restored with a pH-shift to 9.5 which also resulted in ligand field (LF) energy splittings identical to that found for wild type (WT) Dke1 at pH 9.5. In WT Dke1 these LF transitions are shifted up in energy by ~300 cm -1 at pH 9.5 relative to pH 7.0. These data, combined with CD pH titrations which reveal a p K a of ~8.2 for resting WT Dke1 and the Glu98→Gln variant, indicate the deprotonation of a metal ligated water. Together, the kinetic and spectroscopic data reveal a stabilizing effect of Glu98 on the 6C geometry of the metal center, priming it for substrate ligation. Arg80 and Tyr70 are shown to promote O 2 reduction, while Thr107 stabilizes the Fe(II) cofactor.