# Photoinduced Electron Transfer Informs on Pathway Coupling in Flavin-Based Electron Bifurcation

## TL;DR

This paper investigates how electron transfer is controlled in a key enzyme from a heat-loving microbe, revealing insights into how high-energy electrons are managed in a process called electron bifurcation.

## Contribution

The study introduces a novel method combining low-temperature photoexcitation and EPR to probe unstable intermediates in flavin-based electron bifurcation.

## Key findings

- A radical intermediate and its nearby [4Fe-4S] cluster were observed to grow together during cryogenic illumination.
- Photogenerated species remained stable at low temperatures and recombined at higher temperatures.
- The study suggests a potential gating mechanism involving residue movement for reversible electron flow.

## Abstract

Flavin-based electron bifurcation (FBEB) is an enzymatic
mechanism
that generates extremely high-energy electrons to drive unfavorable
chemical reactions. It is utilized by the NADH-dependent ferredoxin:NADP+-oxidoreductase (Nfn) enzyme in hyperthermophile Pyrococcus
furiosus to bifurcate electrons from NADPH into the coupled
low-potential (endergonic) and high-potential (exergonic) pathways.
This process enables P. furiosus to live in harsh
and uninviting environments. Despite its biological importance, the
mechanisms used by Nfn to facilitate exceptional directional control
over short-lived, high-energy electrons and to prevent undesired transfer,
particularly along the low-potential pathway, are still not well understood.
To elucidate how the protein environment contributes to electronic
control in the low-potential pathway, new techniques must be utilized
to probe these unstable intermediates. In this study, we have adapted
low-temperature photoexcitation combined with electron paramagnetic
resonance (EPR) to accumulate the short-lived intermediate and place
it in the context of the other cofactors involved in the low-potential
pathway of Nfn. We observed coincident growth of both the radical
intermediate and its nearby [4Fe-4S] cluster over 4.5 h of illumination
with NADPH at cryogenic temperatures. The photogenerated paramagnetic
species were stable in LN2 storage indefinitely and recombined
when warmed to higher temperatures. The results provide insights into
the electron transfer steps and cofactor interactions along the low
potential pathway, facilitating a more robust mechanistic understanding
of the high-energy events of electron bifurcation. Furthermore, through
comparison of cryogenic and room temperature experiments, a potential
gating step involving the movement of key residues important for the
reversibility of electron flow along this pathway is suggested.