Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Superfamily

Molly J. McBride, Sarah R. Pope, Mrutyunjay A. Nair, Debangsu Sil, Xavier E. Salas-Solá, Carsten Krebs, J. Martin Bollinger, Amie K. Boal

Research output: Chapter in Book/Report/Conference proceedingChapter


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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages32
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics


Dive into the research topics of 'Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Superfamily'. Together they form a unique fingerprint.

Cite this