Two‐Color Valence‐to‐Core X‐ray Emission Spectroscopy Tracks Cofactor Protonation State in a Class I Ribonucleotide Reductase

Abstract

Proton transfer reactions are of central importance to a wide variety of biochemical processes, though determining proton location and monitoring proton transfers in biological systems is often extremely challenging. Herein, we use two-color valence-to-core X-ray emission spectroscopy (VtC XES) to identify protonation events across three oxidation states of the O -activating, radical-initiating manganese-iron heterodinuclear cofactor in a class I-c ribonucleotide reductase. This is the first application of VtC XES to an enzyme intermediate and the first simultaneous measurement of two-color VtC spectra. In contrast to more conventional methods of assessing protonation state, VtC XES is a more direct probe applicable to a wide range of metalloenzyme systems. These data, coupled to insight provided by DFT calculations, allow the inorganic cores of the Mn Fe and Mn Fe states of the enzyme to be assigned as Mn (μ-O) Fe and Mn (μ-O)(μ-OH)Fe , respectively.

The periodic table of ribonucleotide reductases

“…XAS and pulse EPR spectroscopic studies of the Mn IV Fe IV intermediate in class Ic RNR are consistent with a di-μ-oxo bridging structure and a terminal Mn IV hydroxide ligand ( 74 ), suggesting that O 2 inserts between the metal ions to displace the μ-η 1 ,η 2 -bridging Glu ligand, accompanied by deprotonation of the solvent ligand to Mn1. A recent valence-to-core X-ray emission study of this intermediate and the active Mn IV Fe III cofactor confirms this assignment for the Mn IV Fe IV complex ( 75 ). Spectral comparisons show elongation of one Mn-O bond concomitant with contraction of the other upon 1-electron reduction, consistent with protonation of one of the two oxo bridges.…”

Simultaneous two-color X-ray absorption spectroscopy using Laue crystals at an inverse-compton scattering X-ray facility

“…Simultaneous detection of multiple elements has been recently realized by XES employing curved crystals in von Hamos geometry (Gul et al, 2015;Kalinko et al, 2020) and crystal analyzers in Rowland geometry (Finkelstein et al, 2016;Martinie et al, 2018). In these setups, all elements in the sample are illuminated by the same incident X-ray beam.…”

Quantification of Ni–N–O Bond Angles and NO Activation by X-ray Emission Spectroscopy

“…19,20 These significant findings highlighted the value of XES to bioinorganic chemistry in the understanding of enzyme active sites. More recent enzyme studies included two-color valence-to-core (VtC) XES probing both Mn and Fe centers of intermediates in ribonucleotide reductase, 21,22 peptidylglycine monooxygenase, 23 and galactose oxidase. 24 Valence-to-core (VtC) XES probes occupied valence orbitals by detecting relaxation of electrons from ligand-centered 2p and 2s orbitals to the metal-centered 1s orbital (Figure 1A).…”

“…X-ray emission spectroscopy (XES) overcomes some of the limitations of XAS , and is perhaps best known for being able to distinguish C and N from O, as demonstrated in Cr compounds, Mn complexes, a cluster of iron centers, and the enzyme nitrogenase. , These significant findings highlighted the value of XES to bioinorganic chemistry in the understanding of enzyme active sites. More recent enzyme studies included two-color valence-to-core (VtC) XES probing both Mn and Fe centers of intermediates in ribonucleotide reductase, , peptidylglycine monooxygenase, and galactose oxidase …”