Biocatalysis of theophylline oxidation by microbial theophylline oxidase in the presence of non-physiological electron acceptors

Microbial theophylline oxidase (ThOx) is a redox enzyme catalysing 8-hydroxylation of theophylline to form 1,3-dimethyluric acid. In this work, ThOx has been characterized as a fragile haem-containing protein complex composed of several non-covalently bound dynamic domains with molecular weights of around 60 and 210 kDa, and capable of formation of 1.5 MDa assemblies as well. The rate of theophylline oxidation by ThOx with the non-physiological electron acceptor ferricyanide was 0.17 s−1, approaching that with cytochrome c, 0.33 s−1. The apparent catalytic constant depended on the electron acceptor concentration. At concentrations lower than 0.2 mM the reaction did not fit the Michaelis–Menten scheme, and some non-catalytic processes dominated in the overall reaction. The kinetics of ThOx catalysis were also studied at electrodes modified with self-assembled monolayers (SAM) of hydroxyl- and amine-terminated alkanethiols. Different compositions of the SAM provide different orientations of ThOx on these layers. Depending on the orientation of ThOx onto the SAM-modified electrodes, the heterogeneous electron transfer (ET) constant, ks, which characterizes the ET reaction between the electrodes and the haem of ThOx (Eo/ of 87 mV (NHE)) was 0.4 s−1 and 3.2 s−1. Only the low-ET-rate orientation appeared to be productive for the electrocatalytic function of ThOx, giving a reaction similar to that with ferricyanide and cytochrome c. The apparent efficiency of ThOx bioelectrocatalysis in the absence of mediators was substantially lower than that mediated by ferricyanide or cytochrome c. This lower efficiency is consistent with a correspondingly lower amount of ThOx being in direct ET contact with the electrodes and thus involved in electrocatalysis.