Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic CodeCitation formats

  • External authors:
  • Mary Ortmayer
  • Karl Fisher
  • Jaswir Basran
  • Emmanuel M Wolde-Michael
  • J L Ross Anderson
  • Emma L Raven
  • Stephen E J Rigby
  • Anthony P Green

Standard

Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code. / Ortmayer, Mary; Fisher, Karl; Basran, Jaswir; Wolde-Michael, Emmanuel M; Heyes, Derren J; Levy, Colin; Lovelock, Sarah L; Anderson, J L Ross; Raven, Emma L; Hay, Sam; Rigby, Stephen E J; Green, Anthony P.

In: ACS Catalysis, Vol. 10, No. 4, 21.02.2020, p. 2735-2746.

Research output: Contribution to journalArticlepeer-review

Harvard

Ortmayer, M, Fisher, K, Basran, J, Wolde-Michael, EM, Heyes, DJ, Levy, C, Lovelock, SL, Anderson, JLR, Raven, EL, Hay, S, Rigby, SEJ & Green, AP 2020, 'Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code', ACS Catalysis, vol. 10, no. 4, pp. 2735-2746. https://doi.org/10.1021/acscatal.9b05129

APA

Ortmayer, M., Fisher, K., Basran, J., Wolde-Michael, E. M., Heyes, D. J., Levy, C., Lovelock, S. L., Anderson, J. L. R., Raven, E. L., Hay, S., Rigby, S. E. J., & Green, A. P. (2020). Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code. ACS Catalysis, 10(4), 2735-2746. https://doi.org/10.1021/acscatal.9b05129

Vancouver

Ortmayer M, Fisher K, Basran J, Wolde-Michael EM, Heyes DJ, Levy C et al. Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code. ACS Catalysis. 2020 Feb 21;10(4):2735-2746. https://doi.org/10.1021/acscatal.9b05129

Author

Ortmayer, Mary ; Fisher, Karl ; Basran, Jaswir ; Wolde-Michael, Emmanuel M ; Heyes, Derren J ; Levy, Colin ; Lovelock, Sarah L ; Anderson, J L Ross ; Raven, Emma L ; Hay, Sam ; Rigby, Stephen E J ; Green, Anthony P. / Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code. In: ACS Catalysis. 2020 ; Vol. 10, No. 4. pp. 2735-2746.

Bibtex

@article{7ce9324622cf431ba1fbed9a76791c9d,
title = "Rewiring the {"}Push-Pull{"} Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code",
abstract = "Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating Nδ-methyl histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k1) and the reactivity (k2) of compound I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compound II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compound II reactivity, which can be explained by weaker electron donation from the Me-His ligand ({"}the push{"}) affording an electron-deficient ferryl oxygen with reduced proton affinity ({"}the pull{"}). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase {"}the pull{"} by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those observed in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.",
author = "Mary Ortmayer and Karl Fisher and Jaswir Basran and Wolde-Michael, {Emmanuel M} and Heyes, {Derren J} and Colin Levy and Lovelock, {Sarah L} and Anderson, {J L Ross} and Raven, {Emma L} and Sam Hay and Rigby, {Stephen E J} and Green, {Anthony P}",
note = "Copyright {\textcopyright} 2020 American Chemical Society.",
year = "2020",
month = feb,
day = "21",
doi = "10.1021/acscatal.9b05129",
language = "English",
volume = "10",
pages = "2735--2746",
journal = "ACS Catalysis",
issn = "2155-5435",
publisher = "American Chemical Society",
number = "4",

}

RIS

TY - JOUR

T1 - Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code

AU - Ortmayer, Mary

AU - Fisher, Karl

AU - Basran, Jaswir

AU - Wolde-Michael, Emmanuel M

AU - Heyes, Derren J

AU - Levy, Colin

AU - Lovelock, Sarah L

AU - Anderson, J L Ross

AU - Raven, Emma L

AU - Hay, Sam

AU - Rigby, Stephen E J

AU - Green, Anthony P

N1 - Copyright © 2020 American Chemical Society.

PY - 2020/2/21

Y1 - 2020/2/21

N2 - Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating Nδ-methyl histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k1) and the reactivity (k2) of compound I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compound II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compound II reactivity, which can be explained by weaker electron donation from the Me-His ligand ("the push") affording an electron-deficient ferryl oxygen with reduced proton affinity ("the pull"). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase "the pull" by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those observed in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.

AB - Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating Nδ-methyl histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k1) and the reactivity (k2) of compound I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compound II (k3) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compound II reactivity, which can be explained by weaker electron donation from the Me-His ligand ("the push") affording an electron-deficient ferryl oxygen with reduced proton affinity ("the pull"). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase "the pull" by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those observed in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.

U2 - 10.1021/acscatal.9b05129

DO - 10.1021/acscatal.9b05129

M3 - Article

C2 - 32550044

VL - 10

SP - 2735

EP - 2746

JO - ACS Catalysis

JF - ACS Catalysis

SN - 2155-5435

IS - 4

ER -