Mn(II) Binding and Subsequent Oxidation by the Multicopper Oxidase MnxG Investigated by Electron Paramagnetic Resonance Spectroscopy

Abstract

The dynamics of manganese solid formation (as MnOx) by the multicopper oxidase (MCO)-containing Mnx protein complex were examined by electron paramagnetic resonance (EPR) spectroscopy. Continuous-wave (CW) EPR spectra of samples of Mnx, prepared in atmosphere and then reacted with Mn(II) for times ranging from 7 to 600 s, indicate rapid oxidation of the substrate manganese (with two-phase pseudo-first-order kinetics modeled using rate coefficients of: k(1obs) = 0.205 ± 0.001 s(-1) and k(2obs) = 0.019 ± 0.001 s(-1)). This process occurs on approximately the same time scale as in vitro solid MnOx formation when there is a large excess of Mn(II). We also found CW and pulse EPR spectroscopic evidence for at least three classes of Mn(II)-containing species in the reaction mixtures: (i) aqueous Mn(II), (ii) a specifically bound mononuclear Mn(II) ion coordinated to the Mnx complex by one nitrogenous ligand, and (iii) a weakly exchange-coupled dimeric Mn(II) species. These findings provide new insights into the molecular mechanism of manganese mineralization.

Biogenic manganese oxide nanoparticle formation by a multimeric multicopper oxidase Mnx

“…Protein was purified using plasmid constructs and purification methods described previously 30 , 36 , 42 . To isolate untagged protein, the mnxDEFG construct was cloned onto a pTXB1 plasmid (kindly provided by the group of N. Blackburn, Oregon Health & Science University).…”

“…The similar masses of MnxE and MnxF (both ~12 kDa) prevent accurate assignment of subunit stoichiometry unless the molecular mass can be measured at high resolution. Additionally, the many metals bound to Mnx 36 , 41 , 42 give rise to complex spectroscopic signatures 30 , 36 , 42 so it is difficult to investigate individual metal cofactors or binding sites. The enzyme’s fast turnover hinders efforts to capture early and intermediate stages of manganese oxide formation critical for understanding the mechanism of biomineralization 42 .…”

Probing Electron Transfer in the Manganese‐Oxide‐Forming MnxEFG Protein Complex using Fourier Transformed AC Voltammetry: Understanding the Oxidative Priming Effect

“…Mechanistic studies using electron paramagnetic resonance spectroscopy confirmed that the mechanism of the MnxG‐catalysed aerobic oxidation of Mn 2+ is akin to that for oxidation of organic substrates catalysed by smaller multicopper oxidases . In brief, three types of copper cofactors are needed to catalyse four sequential single‐electron oxidations of substrates coupled to the four‐electron reduction of O 2 to H 2 O ,.…”

MopA, the Mn Oxidizing Protein From Erythrobacter sp. SD-21, Requires Heme and NAD+ for Mn(II) Oxidation

“…( Brouwers et al, 2000 ; Ridge et al, 2007 ; Geszvain et al, 2013 ); but the best-characterized enzyme among these is the MnxGEF protein complex derived from Bacillus sp. PL-12 ( Butterfield et al, 2013 ; Tao et al, 2015 ; Butterfield and Tebo, 2017 ; Soldatova et al, 2017a , b ; Tao et al, 2017a , b ). The heterologously expressed and purified MnxGEF complex can perform the two electron oxidation of Mn(II) to Mn(IV) by funneling electrons through mononuclear Type I and trinuclear copper centers to dioxygen.…”