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1
Boston University, Department of Chemistry, Boston, Massachusetts 02215, United States.
2
Carnegie Mellon University, Department of Chemistry, Pittsburgh, Pennsylvania 15213, United States.
3
Massachusetts Institute of Technology, Department of Chemistry, Cambridge, Massachusetts 02139, United States.
4
Massachusetts Institute of Technology, Department of Biology, Cambridge, Massachusetts 02139, United States.
5
Howard Hughes Medical Institute, Cambridge, Massachusetts 02139, United States.
1
Boston University, Department of Chemistry, Boston, Massachusetts 02215, United States.
2
Carnegie Mellon University, Department of Chemistry, Pittsburgh, Pennsylvania 15213, United States.
3
Massachusetts Institute of Technology, Department of Chemistry, Cambridge, Massachusetts 02139, United States.
4
Massachusetts Institute of Technology, Department of Biology, Cambridge, Massachusetts 02139, United States.
5
Howard Hughes Medical Institute, Cambridge, Massachusetts 02139, United States.
BthA is a diheme enzyme that is a member of the bacterial cytochrome c peroxidase superfamily, capable of generating a highly unusual Fe(IV)Fe(IV)═O oxidation state, known to be responsible for long-range oxidative chemistry in the enzyme MauG. Here, we show that installing a canonical Met ligand in lieu of the Tyr found at the heme of MauG associated with electron transfer, results in a construct that yields an unusually stable Fe(IV)═O porphyrin at the peroxidatic heme. This state is spontaneously formed at ambient conditions using either molecular O
2
or H
2
O
2
. The resulting data illustrate how a ferryl iron, with unforeseen stability, may be achieved in biology.
Biophysical characterization of the BthA Y463M variant. (A) Schematic of the 6c heme coordination of BthA and MauG (white) and bCCPs (slate), comparing BthA (PDB ID 6NX0.pdb) and CcpA from
Shewanella oneidensis
(3O5C.pdb). (B) Absorption spectra of aerobically purified (solid) and ascorbate treated (blue) Y463M BthA. (C) Voltammogram of Y463M at pH 7.3, 21˚C, 50 mV/s. Raw voltammogram is shown (black) with the baseline subtracted (black, inset) with fit for the overall signal (green) and individual species fit (green dotted lines, inset). (D) Spectra of 5 μM as-isolated Y463M (solid black line) and then treated with 10x H
2
O
2
(blue)., 50 mM HEPES, pH 7.8.
Compared to the wild-type structure (A; PDB ID: 6NX0), the Y463M mutant (B, PDB ID: 6V59) disrupts axial heme ligation. 2
F
O
–
F
C
composite omit electron density countoured at 1 σ Peptide and heme carbon shown in green and purple, respectively. Oxygen, nitrogen, sulfur, and iron shown in red, blue, yellow, and orange, respectively. Peptide backbone shown in ribbons representation and side chains and hemes shown in stick representation. Side chain α-carbon shown as spheres. Terminal methyl group of Met side chain is not visible in this orientation. 2
F
O
–
F
C
electron density shown in grey and contoured at 1σ.
57
Fe enriched Mössbauer spectra (4.2 K, 45 mT, blue vertical bars) of (A) WT BthA and (B) as-isolated BthA Y463M. (C) Y463M plus 30 equiv sodium ascorbate. (D) C, plus 20 equiv of H
2
O
2
. The black traces overlying the data are simulated sums of species: LS (red dash), HS E/D = 0 (dots), HS E/D = 0.02 (black dash), Fe(IV)=O (red fill). The simulation in C uses the HS species of B, but in larger amounts.
57
Fe enriched Mössbauer spectra (4.2 K, 45 mT, blue vertical bars) of (A) anaerobically purified Y463M BthA, (B) A exposed to O
2
gas for 10 min. The black traces overlying the data are simulated sums using the listed doublet parameters: Fe(II) (blue fill), Fe(II)-CO (green fill), oxy-heme (orange fill), Fe(IV)=O (red fill). The broad magnetic HS species in B are fit with the same species shown in Fig. 3B (black dots and dashed).
Weitz AC, et al.
Inorg Chem. 2020 Jul 20;59(14):10223-10233. doi: 10.1021/acs.inorgchem.0c01349. Epub 2020 Jun 30.
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32602712
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Yukl ET, et al.
Biochemistry. 2011 Apr 12;50(14):2931-8. doi: 10.1021/bi200023n. Epub 2011 Mar 16.
Biochemistry. 2011.
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Rizzolo K, et al.
Nat Commun. 2019 Mar 7;10(1):1101. doi: 10.1038/s41467-019-09020-4.
Nat Commun. 2019.
PMID:
30846684
Free PMC article.
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J Inorg Biochem. 2006 Apr;100(4):551-67. doi: 10.1016/j.jinorgbio.2005.12.008. Epub 2006 Jan 24.
J Inorg Biochem. 2006.
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Jung C, et al.
Arch Biochem Biophys. 2011 Mar 1;507(1):44-55. doi: 10.1016/j.abb.2010.12.029. Epub 2010 Dec 30.
Arch Biochem Biophys. 2011.
PMID:
21195047
Manesis AC, et al.
Biochemistry. 2023 Mar 7;62(5):1082-1092. doi: 10.1021/acs.biochem.3c00021. Epub 2023 Feb 22.
Biochemistry. 2023.
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Free PMC article.
R01 GM077387/GM/NIGMS NIH HHS/United States
HHMI/Howard Hughes Medical Institute/United States
S10 OD021527/OD/NIH HHS/United States
R01 GM110390/GM/NIGMS NIH HHS/United States
R35 GM126982/GM/NIGMS NIH HHS/United States
T32 GM008334/GM/NIGMS NIH HHS/United States
R01 GM072663/GM/NIGMS NIH HHS/United States
P30 GM124165/GM/NIGMS NIH HHS/United States
Show all 8 grants
