EPR characterization of a metal-binding site in RquA

Rhodoquinone (RQ) is a crucial electron carrier involved in anaerobic metabolism across select bacteria, protists, and animal species. Its biosynthesis is catalyzed by the rhodoquinone biosynthesis enzyme (RquA), a methyltransferase-like enzyme that uses S-adenosyl-L-methionine to transfer an amino group, converting ubiquinone (UQ) into RQ. The activity of RquA in vitro is enhanced by the presence of divalent metal cations. To probe the metal dependence of RquA, we characterized its interactions with Mn(II), Co(II), and Zn(II). We found that these metal cations bind to RquA with a 1:1 stoichiometry and that Mn(II) and Co(II) exhibited sub-micromolar binding affinities to RquA. Using Mn(II) as a spectroscopic probe, continuous‑wave electron paramagnetic resonance (EPR) revealed a single Mn(II) species with zero‑field splitting parameters |D| = 540(30) MHz and E/D = 0.30(3). Pulse EPR experiments on natural‑abundance and ¹⁵N‑labeled samples further identified a weakly‑to‑moderately coupled nitrogen ligand, with an isotropic hyperfine coupling constant |a_iso(¹⁵N)| = 2.7(1) MHz. Integrating these data with an AlphaFold3‑derived structural model, we propose a putative binding site for the catalytically required divalent metal cation, providing new insight into the structural basis of RquA function.