| 1. |
- Griese, Julia J., et al.
(författare)
-
Ether cross-link formation in the R2-like ligand-binding oxidase
- 2018
-
Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 23:6, s. 879-886
-
Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of Mn-II and Fe-II in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.
|
|
| 2. |
- Griese, Julia J., et al.
(författare)
-
Structural Basis for Oxygen Activation at a Heterodinuclear Manganese/Iron Cofactor
- 2015
-
Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 290:42, s. 25254-25272
-
Tidskriftsartikel (refereegranskat)abstract
- Two recently discovered groups of prokaryotic di-metal carboxylate proteins harbor a heterodinuclear Mn/Fe cofactor. These are the class Ic ribonucleotide reductase R2 proteins and a group of oxidases that are found predominantly in pathogens and extremophiles, called R2-like ligand-binding oxidases (R2lox). We have recently shown that the Mn/Fe cofactor of R2lox self-assembles from Mn-II and Fe-II in vitro and catalyzes formation of a tyrosine-valine ether cross-link in the protein scaffold (Griese, J. J., Roos, K., Cox, N., Shafaat, H. S., Branca, R.M., Lehtio , J., Graslund, A., Lubitz, W., Siegbahn, P. E., and Hogbom, M. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 1718917194). Here, we present a detailed structural analysis of R2lox in the nonactivated, reduced, and oxidized resting Mn/Fe- and Fe/Fe-bound states, as well as the nonactivated Mn/Mn-bound state. X-ray crystallography and x-ray absorption spectroscopy demonstrate that the active site ligand configuration of R2lox is essentially the same regardless of cofactor composition. Both the Mn/Fe and the diiron cofactor activate oxygen and catalyze formation of the ether cross-link, whereas the dimanganese cluster does not. The structures delineate likely routes for gated oxygen and substrate access to the active site that are controlled by the redox state of the cofactor. These results suggest that oxygen activation proceeds via similar mechanisms at the Mn/Fe and Fe/Fe center and that R2lox proteins might utilize either cofactor in vivo based on metal availability.
|
|
| 3. |
- Kutin, Yury, et al.
(författare)
-
Chemical flexibility of heterobimetallic Mn/Fe cofactors : R2lox and R2c proteins
- 2019
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:48, s. 18372-18386
-
Tidskriftsartikel (refereegranskat)abstract
- A heterobimetallic Mn/Fe cofactor is present in the R2 subunit of class Ic ribonucleotide reductases (R2c) and in R2-like ligand-binding oxidases (R2lox). Although the protein-derived metal ligands are the same in both groups of proteins, the connectivity of the two metal ions and the chemistry each cofactor performs are different: in R2c, a one-electron oxidant, the Mn/Fe dimer is linked by two oxygen bridges (?-oxo/?-hydroxo), whereas in R2lox, a two-electron oxidant, it is linked by a single oxygen bridge (?-hydroxo) and a fatty acid ligand. Here, we identified a second coordination sphere residue that directs the divergent reactivity of the protein scaffold. We found that the residue that directly precedes the N-terminal carboxylate metal ligand is conserved as a glycine within the R2lox group but not in R2c. Substitution of the glycine with leucine converted the resting-state R2lox cofactor to an R2c-like cofactor, a ?-oxo/?-hydroxo?bridged Mn-III/Fe-III dimer. This species has recently been observed as an intermediate of the oxygen activation reaction in WT R2lox, indicating that it is physiologically relevant. Cofactor maturation in R2c and R2lox therefore follows the same pathway, with structural and functional divergence of the two cofactor forms following oxygen activation. We also show that the leucine-substituted variant no longer functions as a two-electron oxidant. Our results reveal that the residue preceding the N-terminal metal ligand directs the cofactor's reactivity toward one- or two-electron redox chemistry, presumably by setting the protonation state of the bridging oxygens and thereby perturbing the redox potential of the Mn ion.
|
|
| 4. |
- Maugeri, Pearson T., et al.
(författare)
-
Driving Protein Conformational Changes with Light : Photoinduced Structural Rearrangement in a Heterobimetallic Oxidase
- 2018
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 140:4, s. 1471-1480
-
Tidskriftsartikel (refereegranskat)abstract
- The heterobimetallic R2lox protein binds both manganese and iron ions in a site-selective fashion and activates oxygen, ultimately performing C-H bond oxidation to generate a tyrosine-valine crosslink near the active site. In this work, we demonstrate that, following assembly, R2lox undergoes photoinduced changes to the active site geometry and metal coordination motif. Through spectroscopic, structural, and mass spectrometric characterization, the photoconverted species is found to consist of a tyrosinate-bound iron center following light-induced decarboxylation of a coordinating glutamate residue and cleavage of the tyrosine-valine cross-link. This process occurs with high quantum efficiencies (Phi = 3%) using violet and near-ultraviolet light, suggesting that the photodecarboxylation is initiated via ligandto-metal charge transfer excitation. Site-directed mutagenesis and structural analysis suggest that the cross-linked tyrosine-162 is the coordinating residue. One primary product is observed following irradiation, indicating potential use of this class of proteins, which contains a putative substrate channel, for controlled photoinduced decarboxylation processes, with relevance for in vivo functionality of R2lox as well as application in environmental remediation.
|
|
| 5. |
- Rapatskiy, Leonid, et al.
(författare)
-
Characterization of Oxygen Bridged Manganese Model Complexes Using Multifrequency (17)O-Hyperfine EPR Spectroscopies and Density Functional Theory
- 2015
-
Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 119:43, s. 13904-13921
-
Tidskriftsartikel (refereegranskat)abstract
- Multifrequency pulsed EPR data are reported for a series of oxygen bridged (μ-oxo/μ-hydroxo) bimetallic manganese complexes where the oxygen is labeled with the magnetically active isotope (17)O (I = 5/2). Two synthetic complexes and two biological metallocofactors are examined: a planar bis-μ-oxo bridged complex and a bent, bis-μ-oxo-μ-carboxylato bridge complex; the dimanganese catalase, which catalyzes the dismutation of H2O2 to H2O and O2, and the recently identified manganese/iron cofactor of the R2lox protein, a homologue of the small subunit of the ribonuclotide reductase enzyme (class 1c). High field (W-band) hyperfine EPR spectroscopies are demonstrated to be ideal methods to characterize the (17)O magnetic interactions, allowing a magnetic fingerprint for the bridging oxygen ligand to be developed. It is shown that the μ-oxo bridge motif displays a small positive isotropic hyperfine coupling constant of about +5 to +7 MHz and an anisotropic/dipolar coupling of -9 MHz. In addition, protonation of the bridge is correlated with an increase of the hyperfine coupling constant. Broken symmetry density functional theory is evaluated as a predictive tool for estimating hyperfine coupling of bridging species. Experimental and theoretical results provide a framework for the characterization of the oxygen bridge in Mn metallocofactor systems, including the water oxidizing cofactor of photosystem II, allowing the substrate/solvent interface to be examined throughout its catalytic cycle.
|
|
