The Methylococcus capsulatus (Bath) Secreted Protein, MopE*, Binds Both Reduced and Oxidized Copper

Thomas Ve,K Kristoffer Andersson,Harald B Jensen,Åsmund K Røhr,Johan R Lillehaug,Rolf-Birger Pedersen,Karina Mathisen,Anne Fjellbirkeland,Ronny Helland,Odd A Karlsen

doi:10.1371/journal.pone.0043146

Abstract

Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A|| = 20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties.

Highlights

Copper is essential for most living organisms, and tight homeostatic regulation of copper acquisition, distribution, and use is generally required
We show by Electron Paramagnetic Resonance (EPR) and X-ray Near Edge Absorption Spectroscopy (XANES) that MopE* binds both reduced and oxidized copper ions
We examined the protein between 4K and 77K without detecting any EPR active signal

Summary

Introduction

Copper is essential for most living organisms, and tight homeostatic regulation of copper acquisition, distribution, and use is generally required. The EPR spectrum of purified MopE* (360 uM) in MOPS buffer did not present any characteristic signal of Cu(II) without added CuCl2, indicating that the copper bound to the purified protein is in the Cu(I) state (Fig. 2B, curve i).

Results

Conclusion