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- Griese, Julia J., et al.
(författare)
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Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein
- 2013
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:43, s. 17189-17194
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Tidskriftsartikel (refereegranskat)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 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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| 4. |
- Shafaat, Hannah S., et al.
(författare)
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Electronic Structural Flexibility of Heterobimetallic Mn/Fe Cofactors : R2lox and R2c Proteins
- 2014
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 136:38, s. 13399-13409
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Tidskriftsartikel (refereegranskat)abstract
- The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Hogbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn-III/Fe-III dimer linked by a mu-hydroxo/bis-mu-carboxylato bridging network. The Mn-III ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2lox(photo)) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn-III/Fe-III cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn-III ion, as best evidenced by a reorientation of its unique Mn-55 hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the mu-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform.
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