Abstract
Nonheme diiron enzymes harness the chemical potential of oxygen to catalyze challenging reactions in biology. In their resting state, these enzymes have a diferrous cofactor that is coordinated by histidine and carboxylate ligands. Upon exposure to oxygen, the cofactor oxidizes to its diferric state forming a peroxo- adduct, capable of catalyzing a wide range of oxidative chemistries such as desaturation and heteroatom oxidation. Despite their versatility and prowess, an emerging subset of nonheme diiron enzymes has inherent cofactor instability making them resistant to structural characterization. This feature is widespread among members of the heme-oxygenase-like diiron oxidase/oxygenase (HDO) superfamily. HDOs have a flexible core structure that remodels upon metal binding. Although ~9600 HDOs have been unearthed, few have undergone functional characterization to date. In this chapter, we describe the methods that have been used to characterize the HDO N-oxygenase, SznF. We demonstrate the overexpression and purification of apo-SznF and methodology specifically designed to aid in obtaining an X-ray structure of holo-SznF. We also describe the characterization of the transient SznF-peroxo-Fe(III)2 complex by stopped-flow absorption and Mössbauer spectroscopies. These studies provide the framework for the characterization of new members of the HDO superfamily.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Nordlund P, Eklund H (1995) Di-iron-carboxylate proteins. Curr Opin Struct Biol 5(6):758–766
Stubbe J (1991) Dinuclear non-heme iron centers: structure and function. Current Opinion in Structural Biology 1(5):788–795. https://doi.org/10.1016/0959-440X(91)90180-2
Jasniewski AJ, Que L Jr (2018) Dioxygen activation by nonheme diiron enzymes: diverse dioxygen adducts, high-valent intermediates, and related model complexes. Chem Rev 118(5):2554–2592. https://doi.org/10.1021/acs.chemrev.7b00457
Rajakovich LJ, Zhang B, McBride MJ, Boal AK, Krebs C, Bollinger JM Jr (2020) Emerging structural and functional diversity in proteins with dioxygen reactive dinuclear transition metal cofactors. In: Liu H-W, Begley TP (eds) Comprehensive natural products III. Elsevier, Amsterdam. https://doi.org/10.1016/B978-0-12-409547-2.14864-4
Schuller DJ, Wilks A, Ortiz de Montellano PR, Poulos TL (1999) Crystal structure of human heme oxygenase-1. Nat Struct Biol 6(9):860–867. https://doi.org/10.1038/12319
McBride MJ, Pope SR, Hu K, Okafor CD, Balskus EP, Bollinger JM Jr, Boal AK (2021) Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF. Proc Natl Acad Sci U S A 118(4). https://doi.org/10.1073/pnas.2015931118
Schwarzenbacher R, Stenner-Liewen F, Liewen H, Robinson H, Yuan H, Bossy-Wetzel E, Reed JC, Liddington RC (2004) Structure of the Chlamydia protein CADD reveals a redox enzyme that modulates host cell apoptosis. J Biol Chem 279(28):29320–29324. https://doi.org/10.1074/jbc.M401268200
Stenner-Liewen F, Liewen H, Zapata JM, Pawlowski K, Godzik A, Reed JC (2002) CADD, a chlamydia protein that interacts with death receptors. J Biol Chem 277(12):9633–9636. https://doi.org/10.1074/jbc.C100693200
Rui Z, Li X, Zhu X, Liu J, Domigan B, Barr I, Cate JH, Zhang W (2014) Microbial biosynthesis of medium-chain 1-alkenes by a nonheme iron oxidase. Proc Natl Acad Sci U S A 111(51):18237–18242. https://doi.org/10.1073/pnas.1419701112
Manley OM, Fan R, Guo Y, Makris TM (2019) Oxidative decarboxylase UndA utilizes a dinuclear iron cofactor. J Am Chem Soc 141(22):8684–8688. https://doi.org/10.1021/jacs.9b02545
Zhang B, Rajakovich LJ, Van Cura D, Blaesi EJ, Mitchell AJ, Tysoe CR, Zhu X, Streit BR, Rui Z, Zhang W, Boal AK, Krebs C, Bollinger JM Jr (2019) Substrate-triggered formation of a peroxo-Fe2(III/III) intermediate during fatty acid decarboxylation by UndA. J Am Chem Soc 141(37):14510–14514. https://doi.org/10.1021/jacs.9b06093
Marchand JA, Neugebauer ME, Ing MC, Lin CI, Pelton JG, Chang MCY (2019) Discovery of a pathway for terminal-alkyne amino acid biosynthesis. Nature 567(7748):420–424. https://doi.org/10.1038/s41586-019-1020-y
Manley OM, Tang H, Xue S, Guo Y, W-c C, Makris TM (2021) BesC initiates C–C cleavage through a substrate-triggered and reactive diferric-peroxo intermediate. Journal of the American Chemical Society 143(50):21416–21424. https://doi.org/10.1021/jacs.1c11109
McBride MJ, Nair MA, Sil D, Slater JW, Neugebauer M, Chang MCY, Boal AK, Krebs C, Bollinger JM (2022) A substrate-triggered μ-peroxodiiron(III) intermediate in the 4-chloro-L-lysine-fragmenting heme-oxygenase-like diiron oxidase (HDO) BesC: substrate dissociation from, and C4 targeting by, the intermediate. bioRxiv 61(8):689–702. https://doi.org/10.1101/2021.12.02.471016
Ng TL, Rohac R, Mitchell AJ, Boal AK, Balskus EP (2019) An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin. Nature 566(7742):94–99. https://doi.org/10.1038/s41586-019-0894-z
McBride M, Sil D, Ng TL, Crooke AM, Kenney GE, Tysoe CR, Zhang B, Balskus EP, Boal AK, Krebs C, Bollinger JM Jr (2020) A peroxodiiron(III/III) intermediate mediating both N-hydroxylation steps in biosynthesis of the N-nitrosourea pharmacophore of streptozotocin by SznF. J Am Chem Soc 142:11818–11828. https://doi.org/10.1021/jacs.0c03431
McBride MJ, Boal AK (2021) SznF, a Metalloenzyme Employed in the Biosynthesis of Streptozotocin. In: Encyclopedia of inorganic and bioinorganic chemistry. Wiley, pp 1–11. https://doi.org/10.1002/9781119951438.eibc2775
Hedges JB, Ryan KS (2019) In vitro reconstitution of the biosynthetic pathway to the nitroimidazole antibiotic azomycin. Angew Chem Int Ed Engl 58(34):11647–11651. https://doi.org/10.1002/anie.201903500
Yun D, García-Serres R, Chicalese BM, An YH, Huynh BH, Bollinger JM Jr (2007) (m-1,2-peroxo)diiron(III/III) complex as a precursor to the diiron(III/IV) intermediate X in the assembly of the iron-radical cofactor of ribonucleotide reductase from mouse. Biochemistry-Us 46(7):1925–1932. https://doi.org/10.1021/bi061717n
McBride MJ, Pope SR, Hu K, Slater JW, Okafor CD, Balskus EP, Bollinger JM Jr, Boal AK (2020) Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF. bioRxiv.:2020.2007.2029.227702. https://doi.org/10.1101/2020.07.29.227702
Acknowledgements
This work was supported by NIH Grants GM119707 (AKB), GM138580 (JMB), and GM127079 (CK).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
McBride, M.J. et al. (2023). Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Superfamily. In: Weinert, E.E. (eds) Oxygen Sensing. Methods in Molecular Biology, vol 2648. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3080-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3080-8_9
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3079-2
Online ISBN: 978-1-0716-3080-8
eBook Packages: Springer Protocols