Mn(II) Oxidation by the Multicopper Oxidase Complex Mnx: A Binuclear Activation Mechanism

Abstract

The bacterial protein complex Mnx contains a multicopper oxidase (MCO) MnxG that, unusually, catalyzes the two-electron oxidation of Mn(II) to MnO biomineral, via a Mn(III) intermediate. Although Mn(III)/Mn(II) and Mn(IV)/Mn(III) reduction potentials are expected to be high, we find a low reduction potential, 0.38 V (vs Normal Hydrogen Electrode, pH 7.8), for the MnxG type 1 Cu, the electron acceptor. Indeed the type 1 Cu is not reduced by Mn(II) in the absence of molecular oxygen, indicating that substrate oxidation requires an activation step. We have investigated the enzyme mechanism via electronic absorption spectroscopy, using chemometric analysis to separate enzyme-catalyzed MnO formation from MnO nanoparticle aging. The nanoparticle aging time course is characteristic of nucleation and particle growth; rates for these processes followed expected dependencies on Mn(II) concentration and temperature, but exhibited different pH optima. The enzymatic time course is sigmoidal, signaling an activation step, prior to turnover. The Mn(II) concentration and pH dependence of a preceding lag phase indicates weak Mn(II) binding. The activation step is enabled by a pK > 8.6 deprotonation, which is assigned to Mn(II)-bound HO; it induces a conformation change (consistent with a high activation energy, 106 kJ/mol) that increases Mn(II) affinity. Mnx activation is proposed to decrease the Mn(III/II) reduction potential below that of type 1 Cu(II/I) by formation of a hydroxide-bridged binuclear complex, Mn(II)(μ-OH)Mn(II), at the substrate site. Turnover is found to depend cooperatively on two Mn(II) and is enabled by a pK 7.6 double deprotonation. It is proposed that turnover produces a Mn(III)(μ-OH)Mn(III) intermediate that proceeds to the enzyme product, likely Mn(IV)(μ-O)Mn(IV) or an oligomer, which subsequently nucleates MnO nanoparticles. We conclude that Mnx exploits manganese polynuclear chemistry in order to facilitate an otherwise difficult oxidation reaction, as well as biomineralization. The mechanism of the Mn(III/IV) conversion step is elucidated in an accompanying paper .

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.…”

“…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. Recent studies on the Mnx complex ( Soldatova et al, 2017a , b ) indicate a unique activation of the enzyme by Mn(II) is required. Binding of a second Mn(II) forms a hydroxide bridged Mn(II)-OH-Mn(II) complex that reduces the high Mn(III)/Mn(II) potential ( E ′ = 1.5 V) to enable electron transfer to the low potential Type I Cu site.…”

Oxidative Formation and Removal of Complexed Mn(III) by Pseudomonas Species

“…The multicopper oxidase (MCO) in Bacillus sp. PL-12 (containing a MnxG subunit with low levels of homology to P. putida GB-1) catalyzes two single one-electron oxidation steps, from Mn(II) to Mn(III) and then Mn(III) to Mn(IV) (Soldatova et al, 2017a , b ). Similarly, animal heme peroxidases produced by A. manganoxydans SI85-9A1, Erythrobacter sp.…”

“…SG-1 (Webb et al, 2005 ; Soldatova et al, 2012 ) and the purified Mnx complex from Bacillus sp. PL-12 (Soldatova et al, 2017a , b ). Given the weak complexation of Mn(III) to PP, we predicted that Mn(III)-citrate would behave in a similar manner.…”

bifA Regulates Biofilm Development of Pseudomonas putida MnB1 as a Primary Response to H2O2 and Mn2+

“…ROS production in microbes during pathological condition is transient and moderate, while their exposure to consequent adverse factors in environments, results in oxidative stress ( Cabiscol et al, 2000 ; Green and Paget, 2004 ; Matallana-Surget et al, 2009 ; Chattopadhyay et al, 2011 ; Murata et al, 2011 ; Chen et al, 2013 ; Imlay, 2013 ). Of note, microbes acquire energy by uptake of organic matter and redox reaction that take place on the transition metal ions in environment, sometimes producing secondary minerals ( Rajkumar and Freitas, 2008 ; Soldatova et al, 2017 ). These physiological adaptation strategies allow microbes to survive in changing environments.…”