Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein
どのようにして、酵素は金属イオンを選択的に取り込むのか。酸素が大事だ
- Julia J. Griesea,
- Katarina Roosb,
- Nicholas Coxc,
- Hannah S. Shafaatc,1,
- Rui M. M. Brancad,
- Janne Lehtiöd,
- Astrid Gräslunda,
- Wolfgang Lubitzc,
- Per E. M. Siegbahnb, and
- Martin Högboma,2
- Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved September 10, 2013 (received for review March 6, 2013)
Significance
Metallocofactors enable enzymes to catalyze difficult reactions that would otherwise not be possible, such as the reduction of oxygen. Nature utilizes a number of different metals, and it is crucial that proteins bind the correct metals to execute their function. Nonetheless, the principles that govern metal specificity in proteins remain poorly understood. Here we use an enzyme that forms a heterodinuclear Mn/Fe cofactor with the same protein ligands in both metal-coordinating positions to study how proteins can differentiate between two such similar metals. We show that the protein is intrinsically capable of site-specific metal discrimination. Surprisingly, specificity is achieved in a stepwise process involving not only fundamental affinity differences, but also chemical maturation upon reaction with molecular oxygen.
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 (MnII < FeII < NiII < CoII < CuII > ZnII). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from MnII and FeII 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 FeII over MnII 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.
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