Similar but Not the Same: First Kinetic and Structural Analyses of a Methanol Dehydrogenase Containing a Europium Ion in the Active Site

Abstract

Since the discovery of the biological relevance of rare earth elements (REEs) for numerous different bacteria, questions concerning the advantages of REEs in the active sites of methanol dehydrogenases (MDHs) over calcium(II) and of why bacteria prefer light REEs have been a subject of debate. Here we report the cultivation and purification of the strictly REE‐dependent methanotrophic bacterium Methylacidiphilum fumariolicum SolV with europium(III), as well as structural and kinetic analyses of the first methanol dehydrogenase incorporating Eu in the active site. Crystal structure determination of the Eu‐MDH demonstrated that overall no major structural changes were induced by conversion to this REE. Circular dichroism (CD) measurements were used to determine optimal conditions for kinetic assays, whereas inductively coupled plasma mass spectrometry (ICP‐MS) showed 70 % incorporation of Eu in the enzyme. Our studies explain why bacterial growth of SolV in the presence of Eu3+ is significantly slower than in the presence of La3+/Ce3+/Pr3+: Eu‐MDH possesses a decreased catalytic efficiency. Although REEs have similar properties, the differences in ionic radii and coordination numbers across the series significantly impact MDH efficiency.

Electrocatalysis of a Europium‐Dependent Bacterial Methanol Dehydrogenase with Its Physiological Electron‐Acceptor Cytochrome cGJ

“…This is in line with the findings from as pectrophotometric horse heart cytochrome c assay,w here only in the presence of MDH, methanol, and cytochrome c GJ was the reduction of as econd cytochrome observed. Cyanide has been included in published MDH assays [11] as ap rotective agent to avoid inhibition by the artificial phenazine ethosulfate mediatora th igh concentrations but this was also not required here when the natural electron acceptor cytochrome c GJ was used. + 200 mV) coincides with that of the cytochrome c GJ redox couple indicating that electrocatalysis is mediated by cytochrome c GJ .…”

“…[11] The higher K M,app value is ar esult of mass transport limitations of thed ialysis membrane. Upon graduala ddition of methanol, the transformation from aq uasi-reversible transient voltammogram (curve a) through an asymmetric peak-shaped response (curvesb !e) and finally to as igmoidal wave above 50 mm methanoliss een with saturation at 230 mm methanol.…”

“…[13] Further,V orholt et al reported that in the uptake of Ln by M. extorquens aT onB related transporter is likely to be involved. [9,11,18] Phenazine ethosulfate and methosulfate have been used as primary electron acceptors for MxaFI-MDHs or XoxF-MDHs in spectrophotometric solution kinetic assays. [15] The reduced( inactive) form of MDH (PQQH 2 )i sr eactivated by two sequential singleelectron transfers to the physiological c-type cytochrome electron acceptor, and PQQH 2 is oxidised back to the quinone via a semiquinone.…”

Biochemical and Structural Characterization of XoxG and XoxJ and Their Roles in Lanthanide‐Dependent Methanol Dehydrogenase Activity

“…The first detailed biochemical and structural characterization of a Ln‐dependent MDH was of XoxF from M. fumariolicum , an organism that requires lanthanides for growth, with La–Nd preferred, although Sm, Eu, and Gd can also support weak growth. Further studies demonstrated that this XoxF had maximal activity in the standard artificial dye‐linked assay (Scheme B) when metallated with Pr and Nd. The activity was ≈30 % lower with La and fell off quickly beyond Nd.…”

Structure and function of the lanthanide-dependent methanol dehydrogenase XoxF from the methanotroph Methylomicrobium buryatense 5GB1C

“…Consistent with the dependence on lanthanides, the only available XoxF structures, those of the Methylacidiphilum ( Ma .) fumariolicum SolV enzyme (SolV-XoxF), reveal Ce(III), La(III), or Eu(III) ions in the active site [12, 18]. …”