Abstract
<jats:p> Redox enzymes are promising biocatalysts for energy conversion and sensing, particularly when immobilized on electrodes to facilitate charge transfer and enhance stability. While they offer the distinct advantages of operating under mild conditions and near-neutral pH, high reaction rates can profoundly alter the immobilized enzyme microenvironment. This requires a deep understanding of the interplay between localized concentration gradients and enzyme activity at the electrocatalytic interface. Here, we elucidate this dynamic coupling between reaction kinetics and local pH at an enzyme-modified electrode through a combined modeling and experimental approach. We present a detailed 2D-axisymmetric finite element model of a graphite microelectrode modified with bilirubin oxidase from Myrothecium verrucaria catalyzing the oxygen reduction reaction via direct electron transfer. The model explicitly accounts for buffer equilibria through ion activities and incorporates experimentally determined pH-dependent kinetic parameters, including turnover rates and Michaelis constants. The simulations were validated experimentally using in situ and operando fluorescence confocal laser scanning microscopy (FCLSM) with the pH-sensitive dye fluorescein in weakly buffered electrolytes. Both numerical and experimental results reveal substantial interfacial alkalinization, with the local pH increasing by more than two units under the weakest buffering conditions. We demonstrate that, beyond directly affecting the turnover constant, this local pH shift governs electrocatalytic activity by increasing the enzyme affinity for O <jats:sub>2</jats:sub> , thereby partially compensating for the loss in activity. The model successfully reproduces cyclic voltammetry and quasi-steady-state chronoamperometry profiles using a single enzyme-coverage-dependent parameter. Furthermore, </jats:p>