Background

Iron-sulfur proteins are of ancient origin, first evolving several billion years ago. They are ubiquitous today, found among all domains of life (Beinert, 2000; Camprubi et al., 2017). These proteins drive almost all major metabolic pathways such as photosynthesis, respiration, nitrogen fixation, methanogenesis, and hydrogen metabolism (Fontecave, 2006; Shaw et al., 2013; Einsle, 2014; Gnandt et al., 2016; Wagner et al., 2016; Lubitz et al., 2019). These proteins function mainly as electron shuttles, routing electrons to the active sites of redox-active enzymes (Johnson et al., 2005; Cordes and Giese, 2009). Furthermore, iron–sulfur proteins are also involved in non-redox functions, such as aconitase, which senses low iron concentrations within cells (Flint and Allen, 1996; Beinert and Kiley, 1999). There are several common cluster compositions, including [2Fe-2S], [3Fe-4S], and [4Fe-4S]. Due to this chemical diversity, they can mediate the transfer of electrons over an extended range between +500 and -680 mV (Kyritsis et al., 1998; Brzoska et al., 2006; Liu et al., 2014).

In this paper, we describe protocols to characterize iron-sulfur proteins using Coiled-Coil Iron-Sulfur (CCIS), an artificial protein [4Fe-4S]-binding protein as a case study (Jagilinki et al., 2020). The CCIS/pET-28a (+) DNA plasmid has been commercially procured (GenScript, USA) [refer toGrzyb et al. (2010) andJagilinki et al. (2020) for the amino acid sequence of the CCIS protein]. The molecular weight of CCIS is 11.6 kDa, and its extinction coefficient at 280 nm is 16,500 (M^-1cm^-1). [4Fe-4S] clusters are highly sensitive to oxygen and require completely anaerobic conditions to study and characterize them (Imlay, 2006). Anaerobic conditions can be maintained in the lab using an anaerobic chamber with 95% nitrogen and 5% hydrogen. The presence of metal ions within the protein enables them to be characterized by several additional biophysical techniques, such as UV-Vis spectroscopy, Electron Paramagnetic Resonance (EPR), and Inductively coupled plasma atomic emission spectroscopy (ICP-AES). The protocols that follow are examples of relevant biochemical and biophysical methods to characterize CCIS and related molecules, specifically UV-visible absorption spectroscopy, Circular Dichroism (CD), thermal denaturation (both secondary structure and [4Fe-4S] cluster), Electron Paramagnetic Resonance (EPR), and Inductively coupled plasma atomic emission spectroscopy (ICP-AES).