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Redox potential increased during the evolution of enzymes degrading recalcitrant lignin
Ayuso-Fernández I, De Lacey AL, Cañada FJ, Ruiz-Dueñas FJ, Martínez AT

Chem. Eur. J., 25: 2708-2712

To investigate how ligninolytic peroxidases acquired the uniquely high redox‐potential they show today, their ancestors were resurrected and characterized. Unfortunately, the transient Compounds I (CI) and II (CII) from peroxide activation of the enzyme resting state (RS) are unstable. Therefore, the reduction potentials (Eº') of the three redox‐couples (CI/RS, CI/CII and CII/RS) were estimated (for the first time in a ligninolytic peroxidase) from equilibria concentrations analyzed by stopped‐flow UV/vis spectroscopy. Interestingly, the Eº' of rate‐limiting CII reduction to RS increased 70 mV from the common peroxidase ancestor to extant lignin peroxidase (LiP), and the same boost was observed for CI/RS and CI/CII, although with higher Eº' values. A straightforward correlation was found between the Eº' and the progressive displacement of the proximal histidine Hε1 shift in NMR spectra, due to higher paramagnetic effect of the heme Fe(III). More interestingly, the Eº' and NMR data also correlate with the evolutionary time, revealing that ancestral peroxidases increased their reduction potential in the evolution to LiP thanks to molecular rearrangements in their heme pocket during the last 400 my.

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