Extended Data Fig. 3: Spectroscopic characterization of MAR metallocofactors.
A) CW X-band EPR spectra (v = 9.38 GHz) of MarHDK. Isolated MarHDK enzyme (135 µM MarH dimer + 70 µM MarDK tetramer) without ATP (dashed), with ATP (solid), and subtraction of resting-state from turnover signal (black) with simulated signals arising from “E1(H)-like” and “E1(H)*-like” species (gray). Spectral intensities are normalized for power. Spectra were collected at T = 15.0 K at the indicated powers. B) Isolated MarHDK enzyme (135 µM MarH dimer + 70 µM MarDK tetramer without ATP (red) and with ATP (green). Isolated MarH protein dimer (435 µM) without ATP (maroon), and with ATP (purple). Associated simulations (gray) for the S = ½ and S = 3/2 signals at 15.0 K, Pµw = 20 mW. * indicates adventitiously bound iron C) Isolated MarHDK enzyme (135 µM MarH dimer + 70 µM MarDK tetramer) at 6 K (blue) and 15 K (red), and isolated MarH protein dimer (435 μM) at 6 K (cyan) and 15 K (maroon) without ATP (dashed) and with ATP (solid) with Pµw = 20 mW. Temperature dependence of S = 3/2 signals in samples without ATP indicate population of ms ± ½ Kramer’s doublet at higher temperature, leading to an assignment of D < 0. * indicates adventitiously bound iron. D) Simulated spin Hamiltonian parameters and corresponding spin quantitation numbers for MarH and MarHDK with and without ATP. a: Values determined from simulation parameters of experimental spectrum with S = ½ and S = 3/2 species. b. Determined from spin quantitation with 186 µM CuII-azurin. EPR experiments were conducted n = 2 times with similar results.
