Daniel Horacio Murgida

Dye-decolorizing peroxidases (DyPs) are heme enzymes with broad substrate scope and increasing biotechnological relevance, yet the structural determinants governing their activation by reactive oxygen species (ROS) remain incompletely understood. Here, we identify dynamic ROS gating as a mechanism controlling activation and catalytic efficiency through a comparative study of two class I DyPs from Bacillus subtilis (BsDyP) and Thermobifida fusca (TfuDyP), together with the BsDyP N244L variant. By combining steady-state kinetics, ROS-selective electroreductive activation assays, UV-vis and resonance Raman spectroscopy, X-ray crystallography, and molecular dynamics simulations, we establish a direct structure-function relationship linking distal heme pocket organization and access-tunnel architecture to enzyme-specific ROS preferences. BsDyP WT is preferentially activated via ^•OH, whereas TfuDyP relies mainly on H₂O₂. Remarkably, the single N244L substitution shifts the catalytic, structural, and dynamical properties of BsDyP toward those of TfuDyP, making both enzymes nearly indistinguishable in ROS usage and catalytic efficiency. This convergence arises from a reorganization of distal hydrogen-bonding networks and loop-mediated reshaping of the access tunnels, which together bias ROS accessibility and lower the barrier for compound I formation. These findings establish ROS gating as the principal determinant of DyP activation and provide a mechanistic framework for tuning ROS selectivity through protein engineering.

Serological diagnostics rely on antigens that are stable, specific, and manufacturable at scale. Full-length viral glycoproteins often fail to meet these criteria due to instability, low expression, or cross-reactivity. Here, we present a modular antigen-display platform that combines rational epitope selection with self-assembling ring-like particles (RLPs) to generate multivalent, solvent-exposed immunoreagents. As a proof of concept, we applied this strategy to glycoprotein E (gE) of Suid herpesvirus 1 (SuHV-1), a key target in DIVA (Differentiating Infected from Vaccinated Animals) diagnostics. Three immunodominant gE regions were identified using IEDB curation and BepiPred-3.0 prediction and grafted onto RLPs to produce a stable ∼18 nm multiepitope construct (RLP-Epi). RLP-Epi assembled homogeneously, remained stable during storage, and was specifically recognized by a commercial anti-gE monoclonal antibody. The construct exhibited reliable performance in both indirect and blocking ELISA formats, with ROC analysis demonstrating high sensitivity and specificity in both configurations. Vaccinated-only sera showed no reactivity against RLP-Epi, confirming DIVA compatibility. These results demonstrate that RLP-based epitope engineering offers a generalizable framework for designing stable, modular diagnostic antigens suitable for rapid adaptation to emerging variants.

Myoglobin, a heme protein abundant in striated muscle cells, plays a crucial role in oxygen delivery to mitochondria. In this study, we combined spectroscopy, rapid kinetics and computer simulations to investigate how the interaction between oxymyoglobin and an outer mitochondrial membrane model - composed of 1:1 POPC:POPE liposomes- impacts oxygen affinity. UV-visible and Resonance Raman spectroscopy studies of metmyoglobin, deoxymyoglobin and oxymyoglobin reveal subtle structural perturbations of the heme binding site, when oxymyoglobin interacts with the model membrane. Stopped-flow kinetic measurements show that these distortions lead to a 2-fold increase in the rate constant for oxygen release. Furthermore, classical and multiscale QM/MM simulations suggest that the increase in the O₂ dissociation rate in the presence of the liposomes is mainly due to proximal effects and heme plane distortions produced by specific interactions between oxymyoglobin and the membrane. Altogether, these results point to a fine-tuning in the active site conformation that facilitates oxygen release from oxymyoglobin upon membrane association, potentially enhancing mitochondrial oxygen availability and energy production in cells.

A dye-decolorizing peroxidase (DyP)-based electrochemical biosensor for hydrogen peroxide (H₂O₂) is developed in miniaturized, disposable, and user-friendly configuration. Wild type and variant DyPs are immobilized on self-assembled monolayer (SAM)-coated and nanostructure-modified screen-printed electrodes (SPEs) to ensure biocompatibility and increase the enzyme loading and hence the biosensor sensitivity. The structure of the enzymes attached to gold and silver nanoparticle (AuNP and AgNP)-modified carbon- and gold-based SPEs (C-SPE and Au-SPE) is monitored by resonance Raman spectroscopy and their electrocatalytic performance toward H₂O₂ by electrochemistry. Among the tested configurations, the wild type DyP/SAM/AuNP/C-SPE construct shows a superior performance, with a linear response range to H₂O₂ from 30 to 475 μM, a sensitivity of 234 ± 9 mA·M^-1·cm^-2, and a limit of detection (LOD) of 3.5 μM, measured in open air conditions. The device is suitable for single-use, on-site measurements of H₂O₂ in air-exposed samples such as physiological fluids. The herein developed biosensor shows a high potential for customization for the detection of other small hydroperoxide substrates and additional improvement of selected traits (e.g., sensitivity, thermal stability, and substrate inhibition properties) due to the easy and well-established production and genetic manipulation of the employed biocatalyst, DyP.