Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein - PubMed (original) (raw)

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Julia J Griese et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving-Williams series (Mn(II) < Fe(II) < Ni(II) < Co(II) < Cu(II) > Zn(II)). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn(II) and Fe(II) in vitro, thus diverging from the Irving-Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe(II) over Mn(II) as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine-valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.

Keywords: EPR spectroscopy; X-ray crystallography; di-metal carboxylate protein; ferritin superfamily; protein metallation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Crystal structures of _Gk_R2loxI. (A) The oxidized Mn/Fe-bound active site at 1.9 Å resolution. (B) The reduced Mn/Fe-bound active site at 1.9 Å resolution. (C) Anomalous difference density at the Fe (gray) and Mn (pink) edges from apoprotein crystals soaked with MnII and FeII in the absence (Upper) or presence (Lower) of oxygen for 3 h, contoured at four electrons per cubic angstrom. At the Fe edge, both Fe and Mn display an anomalous signal. (D) Superposition of the active sites in the metal-free state (at 2.3 Å resolution, colored as in A) and the oxidized Mn/Fe-bound state (transparent gray). Site 2 is preformed before metal binding, whereas site 1 is disordered, with E102 adopting two alternative conformations, and a stretch of four residues including E69 and V72 being invisible in the electron density. (E) mF o -DF c omit electron density for residues Y162 and V72 in apoprotein crystals soaked with MnII and FeII in the absence (Upper) or presence (Lower) of oxygen for 1 h, contoured at 3.0 σ.

Fig. 2.

EPR spectra of _Gk_R2loxI. Labels refer to protein loaded with only Mn (MnII only), 1:1 Mn:Fe under anaerobic conditions (MnII/FeII), and 1:1 Mn to natural abundance Fe (56Fe) or 57Fe under aerobic conditions (MnIII/FeIII). (A) CW X-band EPR spectra. The EPR spectrum of MnII(H2O)6 is overlaid with the Mn-only spectrum for comparison. Asterisks indicate interfering signals from the EPR cavity. (B) Echo-detected, pseudomodulated Q-band EPR spectra. (C) Q-band 55Mn-ENDOR spectra. (D) Q-band 14N-ESEEM spectra, showing the absolute value of the Fourier transform of the time domain measurements. (E) Zero-field Mössbauer spectrum of the MnIII/57FeIII cofactor. The dashed line denotes the fit to the data with an isomer shift of δ = 0.47 mm/s and quadrupole splitting ΔEQ = 0.83 mm/s.

Fig. 3.

Density functional theory study of the mechanism of tyrosine–valine ether cross-link formation. (A) Reaction scheme for the proposed mechanism. (B) Energy profile with key intermediates and rate-limiting transition state.

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Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein - PubMed (original) (raw)