3 research outputs found
Spectroscopic and Computational Comparisons of Thiolate-Ligated Ferric Nonheme Complexes to Cysteine Dioxygenase: Second-Sphere Effects on Substrate (Analogue) Positioning
Parallel spectroscopic and computational studies of iron(III) cysteine dioxygenase (CDO) and synthetic models are presented. The synthetic complexes utilize the ligand tris(4,5-diphenyl-1-methylimidazol-2-yl)phosphine (Ph2TIP), which mimics the facial three-histidine triad of CDO and other thiol dioxygenases. In addition to the previously reported [FeII(CysOEt)(Ph2TIP)]BPh4 (1; CysOEt is the ethyl ester of anionic l-cysteine), the formation and crystallographic characterization of [FeII(2-MTS)(Ph2TIP)]BPh4 (2) is reported, where the methyl 2-thiosalicylate anion (2-MTS) resembles the substrate of 3-mercaptopropionate dioxygenase (MDO). One-electron chemical oxidation of 1 and 2 yields ferric species that bind cyanide and azide anions, which have been used as spectroscopic probes of O2 binding in prior studies of FeIII-CDO. The six-coordinate FeIII-CN and FeIII-N3 adducts are examined with UV–vis absorption, electron paramagnetic resonance (EPR), and resonance Raman (rRaman) spectroscopies. In addition, UV–vis and rRaman studies of cysteine- and cyanide-bound FeIII-CDO are reported for both the wild-type (WT) enzyme and C93G variant, which lacks the Cys-Tyr cross-link that is present in the second coordination sphere of the WT active site. Density functional theory (DFT) and ab initio calculations are employed to provide geometric and electronic structure descriptions of the synthetic and enzymatic FeIII adducts. In particular, it is shown that the complete active space self-consistent field (CASSCF) method, in tandem with n-electron valence state second-order perturbation theory (NEVPT2), is capable of elucidating the structural basis of subtle shifts in EPR g values for low-spin FeIII species. Synopsis
The geometric and electronic structures of thiolate-ligated FeIII complexes of relevance to the active sites of thiol dioxygenases have been elucidated with spectroscopic and computational methods. Data collected for the synthetic models are compared to those previously obtained for the analogous enzymatic species, and newly collected resonance Raman spectra of Cys- and CN-bound FeIII-CDO are presented. The combined enzymatic/synthetic approach reveals that second-sphere residues perturb the positions of substrate (analogues) coordinated to the nonheme iron site of CDO
Characterization by ENDOR Spectroscopy of the Iron–Alkyl Bond in a Synthetic Counterpart of Organometallic Intermediates in Radical SAM Enzymes
Members of the radical S-adenosyl-l-methionine
(SAM) enzyme superfamily initiate a broad spectrum of radical transformations
through reductive cleavage of SAM by a [4Fe–4S]1+ cluster it coordinates to generate the reactive 5′-deoxyadenosyl
radical (5′-dAdo•). However, 5′-dAdo• is not directly liberated for reaction and instead
binds to the unique Fe of the cluster to create the catalytically
competent S = 1/2 organometallic intermediate Ω.
An alternative mode of reductive SAM cleavage, especially seen photochemically,
instead liberates CH3•, which forms the
analogous S = 1/2 organometallic intermediate with
an Fe–CH3 bond, ΩM. The presence
of a covalent Fe–C bond in both structures was established
by the ENDOR observation of 13C and 1H hyperfine
couplings to the alkyl groups that show isotropic components indicative
of Fe–C bond covalency. The synthetic [Fe4S4]3+–CH3 cluster, M-CH3, is a crystallographically characterized
analogue to ΩM that exhibits the same [Fe4S4]3+ cluster state as Ω and ΩM, and thus an analysis of its spectroscopic propertiesand
comparison with those of Ω and ΩMcan
be grounded in its crystal structure. We report cryogenic (2 K) EPR
and 13C/1/2H ENDOR measurements on isotopically
labeled M-CH3. At low temperatures,
the complex exhibits EPR spectra from two distinct conformers/subpopulations.
ENDOR shows that at 2 K, one contains a static methyl, but in the
other, the methyl undergoes rapid tunneling/hopping rotation about
the Fe–CH3 bond. This generates an averaged hyperfine
coupling tensor whose analysis requires an extended treatment of rotational
averaging. The methyl group 13C/1/2H hyperfine
couplings are compared with the corresponding values for Ω and
ΩM
Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC
The MbnBC enzyme complex converts cysteine residues in
a peptide
substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis
of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent
diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III)
cofactor to react with dioxygen for substrate modification. Several
crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone
and in complex with MbnA have been reported, but a mechanistic understanding
requires determination of the oxidation states of the crystallographically
observed Fe ions in the catalytically active Fe(II)Fe(III) state,
along with the site of MbnA binding. Here, we have used electron nuclear
double resonance (ENDOR) spectroscopy to determine such structural
and electronic properties of the active site, in particular, the mode
of substrate binding to the MV state, information not accessible by
X-ray crystallography alone. The oxidation states of the two Fe ions
were determined by 15N ENDOR analysis. The presence and
locations of both bridging and terminal exogenous solvent ligands
were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates
that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom
of its N-terminal modifiable cysteine residue, with
displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR
in studying MVDOs, provide a molecular picture of the initial steps
in Mbn biosynthesis