skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate–dependent nonheme ferrous enzymes

Abstract

Determining the requirements for efficient oxygen (O2) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)–dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O2) and a sequential mechanism (cofactor binding and reaction with O2precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Interestingly, spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the FeII), αKG binding to FeII-DAOCS results in ~45% five-coordinate sites that selectively react with O2relative to the remaining six-coordinate sites. However, this reaction produces an FeIIIspecies that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to FeII-DAOCS produces five-coordinate sites that rapidly react with O2to form an FeIV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes.

Authors:
 [1];  [1];  [1]; ORCiD logo [2]; ORCiD logo [3];  [3];  [4];  [2]; ORCiD logo [2]
  1. Stanford Univ., CA (United States)
  2. Stanford Univ., CA (United States); SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
  3. Technical Univ. of Denmark, Lyngby (Denmark)
  4. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
National Institutes of Health (NIH); USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institute of General Medical Sciences (NIGMS); Independent Research Fund Denmark
OSTI Identifier:
1608322
Grant/Contract Number:  
AC02-76SF00515; GM 40392; P41GM103393; DFF-6108-00247
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 117; Journal Issue: 10; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; oxygen activation; concerted mechanism; metalloenzymes; oxygenase chemistry

Citation Formats

Goudarzi, Serra, Iyer, Shyam R., Babicz, Jr., Jeffrey T., Yan, James J., Peters, Günther H. J., Christensen, Hans E. M., Hedman, Britt, Hodgson, Keith O., and Solomon, Edward I. Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate–dependent nonheme ferrous enzymes. United States: N. p., 2020. Web. https://doi.org/10.1073/pnas.1922484117.
Goudarzi, Serra, Iyer, Shyam R., Babicz, Jr., Jeffrey T., Yan, James J., Peters, Günther H. J., Christensen, Hans E. M., Hedman, Britt, Hodgson, Keith O., & Solomon, Edward I. Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate–dependent nonheme ferrous enzymes. United States. https://doi.org/10.1073/pnas.1922484117
Goudarzi, Serra, Iyer, Shyam R., Babicz, Jr., Jeffrey T., Yan, James J., Peters, Günther H. J., Christensen, Hans E. M., Hedman, Britt, Hodgson, Keith O., and Solomon, Edward I. Mon . "Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate–dependent nonheme ferrous enzymes". United States. https://doi.org/10.1073/pnas.1922484117. https://www.osti.gov/servlets/purl/1608322.
@article{osti_1608322,
title = {Evaluation of a concerted vs. sequential oxygen activation mechanism in α-ketoglutarate–dependent nonheme ferrous enzymes},
author = {Goudarzi, Serra and Iyer, Shyam R. and Babicz, Jr., Jeffrey T. and Yan, James J. and Peters, Günther H. J. and Christensen, Hans E. M. and Hedman, Britt and Hodgson, Keith O. and Solomon, Edward I.},
abstractNote = {Determining the requirements for efficient oxygen (O2) activation is key to understanding how enzymes maintain efficacy and mitigate unproductive, often detrimental reactivity. For the α-ketoglutarate (αKG)–dependent nonheme iron enzymes, both a concerted mechanism (both cofactor and substrate binding prior to reaction with O2) and a sequential mechanism (cofactor binding and reaction with O2precede substrate binding) have been proposed. Deacetoxycephalosporin C synthase (DAOCS) is an αKG-dependent nonheme iron enzyme for which both of these mechanisms have been invoked to generate an intermediate that catalyzes oxidative ring expansion of penicillin substrates in cephalosporin biosynthesis. Interestingly, spectroscopy shows that, in contrast to other αKG-dependent enzymes (which are six coordinate when only αKG is bound to the FeII), αKG binding to FeII-DAOCS results in ~45% five-coordinate sites that selectively react with O2relative to the remaining six-coordinate sites. However, this reaction produces an FeIIIspecies that does not catalyze productive ring expansion. Alternatively, simultaneous αKG and substrate binding to FeII-DAOCS produces five-coordinate sites that rapidly react with O2to form an FeIV=O intermediate that then reacts with substrate to produce cephalosporin product. These results demonstrate that the concerted mechanism is operative in DAOCS and by extension, other nonheme iron enzymes.},
doi = {10.1073/pnas.1922484117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 10,
volume = 117,
place = {United States},
year = {2020},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Save / Share:

Works referenced in this record:

Two Distinct Mechanisms for C–C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of α-Heteroatom Assistance
journal, April 2018

  • Dunham, Noah P.; Chang, Wei-chen; Mitchell, Andrew J.
  • Journal of the American Chemical Society, Vol. 140, Issue 23
  • DOI: 10.1021/jacs.8b01933

Cephalosporin biosynthesis: A branched pathway sensitive to an isotope effect
journal, December 1991


Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme
journal, July 2005

  • Higgins, Luke J.; Yan, Feng; Liu, Pinghua
  • Nature, Vol. 437, Issue 7060
  • DOI: 10.1038/nature03924

The roles of clavaminic acid and proclavaminic acid in clavulanic acid biosynthesis
journal, January 1987

  • Elson, Stephen W.; Baggaley, Keith H.; Gillett, Janet
  • Journal of the Chemical Society, Chemical Communications, Issue 22
  • DOI: 10.1039/c39870001739

Tetrahydropterin-Dependent Amino Acid Hydroxylases
journal, June 1999


Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIVboxHO species
journal, February 2010

  • Grzyska, P. K.; Appelman, E. H.; Hausinger, R. P.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 9
  • DOI: 10.1073/pnas.0911565107

Circular Dichroism and Magnetic Circular Dichroism Spectroscopic Studies of the Non-Heme Ferrous Active Site in Clavaminate Synthase and Its Interaction with α-Ketoglutarate Cosubstrate
journal, February 1998

  • Pavel, Elizabeth G.; Zhou, Jing; Busby, Robert W.
  • Journal of the American Chemical Society, Vol. 120, Issue 4
  • DOI: 10.1021/ja972408a

Magnetic Circular Dichroism Spectroscopic Studies of Mononuclear Non-Heme Ferrous Model Complexes. Correlation of Excited- and Ground-State Electronic Structure with Geometry
journal, April 1998

  • Pavel, Elizabeth G.; Kitajima, Nobumasa; Solomon, Edward I.
  • Journal of the American Chemical Society, Vol. 120, Issue 16
  • DOI: 10.1021/ja973735l

Evidence for Modulation of Oxygen Rebound Rate in Control of Outcome by Iron(II)- and 2-Oxoglutarate-Dependent Oxygenases
journal, September 2019

  • Pan, Juan; Wenger, Eliott S.; Matthews, Megan L.
  • Journal of the American Chemical Society, Vol. 141, Issue 38
  • DOI: 10.1021/jacs.9b06689

Structure of Factor-inhibiting Hypoxia-inducible Factor (HIF) Reveals Mechanism of Oxidative Modification of HIF-1α
journal, November 2002

  • Elkins, Jonathan M.; Hewitson, Kirsty S.; McNeill, Luke A.
  • Journal of Biological Chemistry, Vol. 278, Issue 3
  • DOI: 10.1074/jbc.C200644200

Substrate Binding to the α-Ketoglutarate-Dependent Non-Heme Iron Enzyme Clavaminate Synthase 2:  Coupling Mechanism of Oxidative Decarboxylation and Hydroxylation
journal, December 1998

  • Zhou, Jing; Gunsior, Michele; Bachmann, Brian O.
  • Journal of the American Chemical Society, Vol. 120, Issue 51
  • DOI: 10.1021/ja983534x

Kinetic and crystallographic studies on deacetoxycephalosporin C synthase (DAOCS)
journal, May 2001

  • Lee, Hwei-Jen; Lloyd, Matthew D.; Harlos, Karl
  • Journal of Molecular Biology, Vol. 308, Issue 5
  • DOI: 10.1006/jmbi.2001.4649

The biosynthesis of penicillins and cephalosporins
journal, January 1988

  • Baldwin, J. E.; Abraham, Edward
  • Natural Product Reports, Vol. 5, Issue 2
  • DOI: 10.1039/np9880500129

Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases
journal, July 2015

  • Martinez, Salette; Hausinger, Robert P.
  • Journal of Biological Chemistry, Vol. 290, Issue 34
  • DOI: 10.1074/jbc.R115.648691

The structural basis of cephalosporin formation in a mononuclear ferrous enzyme
journal, December 2003

  • Valegård, Karin; van Scheltinga, Anke C. Terwisscha; Dubus, Alain
  • Nature Structural & Molecular Biology, Vol. 11, Issue 1
  • DOI: 10.1038/nsmb712

α-Amine Desaturation of d -Arginine by the Iron(II)- and 2-(Oxo)glutarate-Dependent l -Arginine 3-Hydroxylase, VioC
journal, October 2018


Oxidative dealkylation DNA repair mediated by the mononuclear non-heme iron AlkB proteins
journal, April 2006


Structure of a cephalosporin synthase
journal, August 1998

  • Valegård, Karin; van Scheltinga, Anke C. Terwisscha; Lloyd, Matthew D.
  • Nature, Vol. 394, Issue 6695
  • DOI: 10.1038/29575

Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites
journal, February 2003


Aberrant activity of the DNA repair enzyme AlkB
journal, May 2004


O 2 Activation by Nonheme Fe II α-Ketoglutarate-Dependent Enzyme Variants: Elucidating the Role of the Facial Triad Carboxylate in FIH
journal, August 2018

  • Iyer, Shyam R.; Chaplin, Vanessa D.; Knapp, Michael J.
  • Journal of the American Chemical Society, Vol. 140, Issue 37
  • DOI: 10.1021/jacs.8b07277

Spectroscopy and Kinetics of Wild-Type and Mutant Tyrosine Hydroxylase: Mechanistic Insight into O 2 Activation
journal, June 2009

  • Chow, Marina S.; Eser, Bekir E.; Wilson, Samuel A.
  • Journal of the American Chemical Society, Vol. 131, Issue 22
  • DOI: 10.1021/ja810080c

Autocatalysed oxidative modifications to 2-oxoglutarate dependent oxygenases: Oxidative modifications to 2OG oxygenases
journal, February 2012


Spectroscopic and Electronic Structure Studies of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase:  O 2 Reactivity of the Non-Heme Ferrous Site in Extradiol Dioxygenases
journal, September 2003

  • Davis, Mindy I.; Wasinger, Erik C.; Decker, Andrea
  • Journal of the American Chemical Society, Vol. 125, Issue 37
  • DOI: 10.1021/ja029746i

CD and MCD of CytC3 and Taurine Dioxygenase:  Role of the Facial Triad in α-KG-Dependent Oxygenases
journal, October 2007

  • Neidig, Michael L.; Brown, Christina D.; Light, Kenneth M.
  • Journal of the American Chemical Society, Vol. 129, Issue 46
  • DOI: 10.1021/ja074557r

Kinetic Dissection of the Catalytic Mechanism of Taurine:α-Ketoglutarate Dioxygenase (TauD) from Escherichia coli
journal, June 2005

  • Price, John C.; Barr, Eric W.; Hoffart, Lee M.
  • Biochemistry, Vol. 44, Issue 22
  • DOI: 10.1021/bi050227c

Utilization of Hydrogen Bonds To Stabilize M−O(H) Units:  Synthesis and Properties of Monomeric Iron and Manganese Complexes with Terminal Oxo and Hydroxo Ligands
journal, February 2004

  • MacBeth, Cora E.; Gupta, Rajeev; Mitchell-Koch, Katie R.
  • Journal of the American Chemical Society, Vol. 126, Issue 8
  • DOI: 10.1021/ja0305151

Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium.
journal, January 1987


Characterization of Terminal Iron(III)–Oxo and Iron(III)–Hydroxo Complexes Derived from O 2 Activation
journal, November 2019


Orthric Rieske dioxygenases for degrading mixtures of 2,4-dinitrotoluene/naphthalene and 2-amino-4,6-dinitrotoluene/4-amino-2,6-dinitrotoluene
journal, December 2006

  • Keenan, Brendan G.; Wood, Thomas K.
  • Applied Microbiology and Biotechnology, Vol. 73, Issue 4
  • DOI: 10.1007/s00253-006-0538-8

Spectroscopic Studies of the Mononuclear Non-Heme Fe II Enzyme FIH: Second-Sphere Contributions to Reactivity
journal, June 2013

  • Light, Kenneth M.; Hangasky, John A.; Knapp, Michael J.
  • Journal of the American Chemical Society, Vol. 135, Issue 26
  • DOI: 10.1021/ja312571m

Geometric and Electronic Structure/Function Correlations in Non-Heme Iron Enzymes
journal, January 2000

  • Solomon, Edward I.; Brunold, Thomas C.; Davis, Mindy I.
  • Chemical Reviews, Vol. 100, Issue 1
  • DOI: 10.1021/cr9900275

Variable-temperature variable-field magnetic circular dichroism studies of the iron(II) active site in metapyrocatechase: implications for the molecular mechanism of extradiol dioxygenases
journal, May 1991

  • Mabrouk, Patricia A.; Orville, Allen M.; Lipscomb, John D.
  • Journal of the American Chemical Society, Vol. 113, Issue 11
  • DOI: 10.1021/ja00011a001

Studies on the active site of deacetoxycephalosporin C synthase
journal, April 1999

  • Lloyd, Matthew D.; Lee, Hwei-Jen; Harlos, Karl
  • Journal of Molecular Biology, Vol. 287, Issue 5
  • DOI: 10.1006/jmbi.1999.2594

Geometric and Electronic Structure Contributions to Function in Non-heme Iron Enzymes
journal, July 2013

  • Solomon, Edward I.; Light, Kenneth M.; Liu, Lei V.
  • Accounts of Chemical Research, Vol. 46, Issue 11
  • DOI: 10.1021/ar400149m

Stereochemical fate of chiral methyl of valine in the ring expansion of penicillin N to deacetoxycephalosporin C
journal, August 1985

  • Townsend, Craig A.; Theis, Alan B.; Neese, A. Scott
  • Journal of the American Chemical Society, Vol. 107, Issue 16
  • DOI: 10.1021/ja00302a026

Measurement of g Values in the Electron Spin Resonance Spectra of Free Radicals
journal, December 1965

  • Segal, Bernice G.; Kaplan, Michael; Fraenkel, George K.
  • The Journal of Chemical Physics, Vol. 43, Issue 12
  • DOI: 10.1063/1.1696676

Studies on Deacetoxycephalosporin C Synthase Support a Consensus Mechanism for 2-Oxoglutarate Dependent Oxygenases
journal, April 2014

  • Tarhonskaya, Hanna; Szöllössi, Andrea; Leung, Ivanhoe K. H.
  • Biochemistry, Vol. 53, Issue 15
  • DOI: 10.1021/bi500086p

Spectroscopic Evidence for a High-Spin Br-Fe(IV)-Oxo Intermediate in the α-Ketoglutarate-Dependent Halogenase CytC3 from Streptomyces
journal, November 2007

  • Galonić Fujimori, Danica; Barr, Eric W.; Matthews, Megan L.
  • Journal of the American Chemical Society, Vol. 129, Issue 44
  • DOI: 10.1021/ja076454e

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase
journal, May 2001

  • Dubus, A.; Lloyd, M. D.; Lee, H. -J.
  • Cellular and Molecular Life Sciences, Vol. 58, Issue 5
  • DOI: 10.1007/PL00000904

X-ray Absorption Spectroscopy Structural Investigation of Early Intermediates in the Mechanism of DNA Repair by Human ABH2
journal, June 2011

  • Giri, Nitai Charan; Sun, Hong; Chen, Haobin
  • Biochemistry, Vol. 50, Issue 22
  • DOI: 10.1021/bi101668x

O 2 - and α-Ketoglutarate-Dependent Tyrosyl Radical Formation in TauD, an α-Keto Acid-Dependent Non-Heme Iron Dioxygenase
journal, February 2003

  • Ryle, Matthew J.; Liu, Aimin; Muthukumaran, Rajendra Bose
  • Biochemistry, Vol. 42, Issue 7
  • DOI: 10.1021/bi026832m

Alternative Reactivity of an α-Ketoglutarate-Dependent Iron(II) Oxygenase:  Enzyme Self-Hydroxylation
journal, May 2001

  • Liu, Aimin; Ho, Raymond Y. N.; Que, Lawrence
  • Journal of the American Chemical Society, Vol. 123, Issue 21
  • DOI: 10.1021/ja005879x

Dioxygen activation by nonheme iron enzymes with the 2-His-1-carboxylate facial triad that generate high-valent oxoiron oxidants
journal, January 2017

  • Kal, Subhasree; Que, Lawrence
  • JBIC Journal of Biological Inorganic Chemistry, Vol. 22, Issue 2-3
  • DOI: 10.1007/s00775-016-1431-2

Direct spectroscopic detection of a C-H-cleaving high-spin Fe(IV) complex in a prolyl-4-hydroxylase
journal, September 2006

  • Hoffart, L. M.; Barr, E. W.; Guyer, R. B.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 40
  • DOI: 10.1073/pnas.0604005103

Electron Paramagnetic Resonance of Some Sulfur and Selenium Compounds
journal, October 1964

  • Windle, J. J.; Wiersema, A. K.; Tappel, A. L.
  • The Journal of Chemical Physics, Vol. 41, Issue 7
  • DOI: 10.1063/1.1726194

Substrate-Triggered Formation and Remarkable Stability of the C−H Bond-Cleaving Chloroferryl Intermediate in the Aliphatic Halogenase, SyrB2
journal, May 2009

  • Matthews, Megan L.; Krest, Courtney M.; Barr, Eric W.
  • Biochemistry, Vol. 48, Issue 20
  • DOI: 10.1021/bi900109z

X-ray Crystal Structure of Escherichia coli Taurine/α-Ketoglutarate Dioxygenase Complexed to Ferrous Iron and Substrates ,
journal, April 2002

  • Elkins, Jonathan M.; Ryle, Matthew J.; Clifton, Ian J.
  • Biochemistry, Vol. 41, Issue 16
  • DOI: 10.1021/bi016014e

O 2 Activation by Non-Heme Iron Enzymes
journal, November 2016