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Second Coordination Sphere Effects on the Mechanistic Pathways for Dioxygen Activation by a Ferritin: Involvement of a Tyr Radical and the Identification of a Cation Binding Site.

ABSTRACT: Ferritins are ubiquitous diiron enzymes involved in iron(II) detoxification and oxidative stress responses and can act as metabolic iron stores. The overall reaction mechanisms of ferritin enzymes are still unclear, particularly concerning the role of the conserved, near catalytic center Tyr residue. Thus, we carried out a computational study of a ferritin using a large cluster model of well over 300 atoms including its first- and second-coordination sphere. The calculations reveal important insight into the structure and reactivity of ferritins. Specifically, the active site Tyr residue delivers a proton and electron in the catalytic cycle prior to iron(II) oxidation. In addition, the calculations highlight a likely cation binding site at Asp₆₅ , which through long-range electrostatic interactions, influences the electronic configuration and charge distributions of the metal center. The results are consistent with experimental observations but reveal novel detail of early mechanistic steps that lead to an unusual mixed-valent iron(III)-iron(II) center.

SUBMITTER: Yeh CG

PROVIDER: S-EPMC9401865 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Second Coordination Sphere Effects on the Mechanistic Pathways for Dioxygen Activation by a Ferritin: Involvement of a Tyr Radical and the Identification of a Cation Binding Site.

Yeh Chieh-Chih George CG Mokkawes Thirakorn T Bradley Justin M JM Le Brun Nick E NE de Visser Sam P SP

Chembiochem : a European journal of chemical biology 20220523 13

Ferritins are ubiquitous diiron enzymes involved in iron(II) detoxification and oxidative stress responses and can act as metabolic iron stores. The overall reaction mechanisms of ferritin enzymes are still unclear, particularly concerning the role of the conserved, near catalytic center Tyr residue. Thus, we carried out a computational study of a ferritin using a large cluster model of well over 300 atoms including its first- and second-coordination sphere. The calculations reveal important ins ...[more]

PMID: 35510795

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Project description:The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn(803) within the ?-subunit of the hypoxia inducible factor (HIF). FIH is an ?-ketoglutatrate (?KG)-dependent, non-heme Fe(II) dioxygenase, in which Fe(II) is coordinated by a (His(2)Asp) facial triad, ?KG, and H(2)O. Hydrogen bonding among the facial triad, the HIF-Asn(803) side chain, and various second-sphere residues suggests a functional role for the second coordination sphere in tuning the chemistry of the Fe(II) center. Point mutants of FIH were prepared to test the functional role of the ?KG-centered (Asn(205) and Asn(294)) or HIF-Asn(803)-centered (Arg(238) and Gln(239)) second-sphere residues. The second sphere was tested for local effects on priming Fe(II) to react with O(2), oxidative decarboxylation, and substrate positioning. Steady-sate kinetics were used to test for overall catalytic effects; autohydroxylation rates were used to test for priming and positioning, and electronic spectroscopy was used to assess the primary coordination sphere and the electrophilicity of ?KG. Asn(205) ? Ala and Asn(294) ? Ala mutants exhibited diminished rates of steady-state turnover, while minimally affecting autohydroxylation, consistent with impaired oxidative decarboxylation. Blue-shifted metal to ligand charge transfer transitions for (Fe+?KG)FIH indicated that these point mutations destabilized the ?* orbitals of ?KG, further supporting a slowed rate of oxidative decarboxylation. The Arg(238) ? Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; the Arg(238) ? Met mutant was capable of O(2) activation for the autohydroxylation reaction. The Gln(239) ? Asn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to the Gln(239) ? Asn mutant stimulated autohydroxylation, it is more likely that this point mutant simply mispositions the HIF-Asn(803) side chain. This work combines kinetics and spectroscopy to show that these second-sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O(2), and positioning HIF-Asn(803).

Dataset Information

Second Coordination Sphere Effects on the Mechanistic Pathways for Dioxygen Activation by a Ferritin: Involvement of a Tyr Radical and the Identification of a Cation Binding Site.

Publications

Second Coordination Sphere Effects on the Mechanistic Pathways for Dioxygen Activation by a Ferritin: Involvement of a Tyr Radical and the Identification of a Cation Binding Site.

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