Disruption of the H-bond network in the main access channel of catalase-peroxidase modulates enthalpy and entropy of Fe(III) reduction.

Catalase-peroxidases are the only heme peroxidases with substantial hydrogen peroxide dismutation activity. In order to understand the role of the redox chemistry in their bifunctional activity, catalatically-active and inactive mutant proteins have been probed in spectroelectrochemical experiments. In detail, wild-type KatG from Synechocystis has been compared with variants with (i) disrupted KatG-typical adduct (Trp122-Tyr249-Met275), (ii) mutation of the catalytic distal His123-Arg119 pair, and (iii) altered accessibility to the heme cavity (Asp152, Ser335) and modified charge at the substrate channel entrance (Glu253). A valuable insight into the mechanism of reduction potential (E degrees ') modulation in KatG has been obtained from the parameterization of the corresponding enthalpic and entropic components, determined from the analysis of the temperature dependence of E degrees '. Moreover, model structures of ferric and ferrous Synechocystis KatG have been computed and used as reference to analyze and discuss the experimental data. The results, discussed by reference to published resonance Raman data on the strength of the proximal iron-imidazole bond and catalytic properties, demonstrate that E degrees ' of the Fe(III)/Fe(II) couple is not strongly correlated with the bifunctional activity. Besides the importance of an intact Trp-Tyr-Met adduct, it is the architecture of the long and constricted main channel that distinguishes KatGs from monofunctional peroxidases. An ordered matrix of oriented water dipoles is important for H(2)O(2) oxidation. Its disruption results in modification of enthalpic and entropic contributions to E degrees ' that reflect reduction-induced changes in polarity, electrostatics, continuity and accessibility of solvent to the metal center as well as alterations in solvent reorganization.