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

Catalase–peroxidases are the only heme peroxidases with substantial hydrogen peroxide dismutation 28activity. In order to understand the role of the redox chemistry in their bifunctional activity, catalatically- 29active and inactive mutant proteins have been probed in spectroelectrochemical experiments. In detail, wild- 30type KatG from Synechocystis has been compared with variants with (i) disrupted KatG-typical adduct 31(Trp122-Tyr249-Met275), (ii) mutation of the catalytic distal His123-Arg119 pair, and (iii) altered 32accessibility to the heme cavity (Asp152, Ser335) and modified charge at the substrate channel entrance 33(Glu253). A valuable insight into the mechanism of reduction potential (E°′) modulation in KatG has been 34obtained from the parameterization of the corresponding enthalpic and entropic components, determined 35from the analysis of the temperature dependence of E°′. Moreover, model structures of ferric and ferrous 36Synechocystis KatG have been computed and used as reference to analyze and discuss the experimental data. 37The results, discussed by reference to published resonance Raman data on the strength of the proximal iron- 38imidazole bond and catalytic properties, demonstrate that E°′ of the Fe(III)/Fe(II) couple is not strongly 39correlated with the bifunctional activity. Besides the importance of an intact Trp-Tyr-Met adduct, it is the 40architecture of the long and constricted main channel that distinguishes KatGs from monofunctional 41peroxidases. An ordered matrix of oriented water dipoles is important for H2O2 oxidation. Its disruption 42results in modification of enthalpic and entropic contributions to E°′ that reflect reduction-induced changes 43in polarity, electrostatics, continuity and accessibility of solvent to the metal center as well as alterations in 44solvent reorganization.