Lichtenstein, B. R. | bioRxiv Science

bioRxiv2026

Porphyrin driven redox tuning in structurally defined de novo heme proteins

Designing redox proteins with predictable and tuneable electron transfer properties is a major goal in de novo bioenergetics. Here we show that replacing heme B with a series of structurally conservative non-natural metalloporphyrins enables broad modulation of redox potentials over 400 mV in the de novo designed monoheme m4D2 and diheme 4D2 T19D. The non-natural porphyrins bind with high affinity and do not compromise either the heme binding site or global protein structure, as evidenced by X-ray crystallography and NMR spectroscopy. We also report the native-like NMR structure of m4D2 loaded with the non-natural and symmetric iron 2,4-dimethyldeuteroporphyrin IX, confirming our modular approach to tetrahelical redox protein design. This work establishes a versatile platform for constructing tuneable electron carriers for engineered bioenergetic pathways and bioelectronic applications.

biochemistry↗

bioRxiv2025

Engineering surface electrostatics affords control over morphological preference, synergy, and activity in polymer degrading enzymes

The biocatalytic recycling of plastics, such as polyethylene terephthalate (PET), promises a sustainable alternative to our present open-loop cycles. Engineering of PET-hydrolases for this purpose has focused on improving activity near the glass-transition temperature of the polymer by increasing their thermostability, neglecting other features of the protein-polymer system that affect enzymatic activity. Here, we isolate the effect of electrostatics on the activity of a thermophilic PETase by rationally redesigning its surface charge, while preserving its thermodynamic properties. The enzyme variant, SfInv, shows orders of magnitude improvements in binding affinity and in activity towards untreated plastic films, with inverted morphological preference. When combined, the wildtype enzyme and SfInv act synergistically, revealing an entirely new mechanism for cooperative activities driven by complimentary electrostatic interactions at the PET surface. These findings highlight unexplored avenues in improving PETase function through the control of morphological preference or introduction of protein cooperativity by exploiting protein electrostatics.

biochemistry↗