Robust Production, Crystallization, Structure Determination, and Analysis of [Fe–S] Proteins: Uncovering Control of Electron Shuttling and Gating in the Respiratory Metabolism of Molybdopterin Guanine Dinucleotide Enzymes

Abstract

[Fe–S] clusters are essential cofactors in all domains of life. They play many biological roles due to their unique abilities for electron transfer and conformational control. Yet, producing and analyzing Fe–S proteins can be difficult and even misleading if not done anaerobically. Due to unique redox properties of [Fe–S] clusters and their oxygen sensitivity, they pose multiple challenges and can lose enzymatic activity or cause their component proteins to be structurally disordered due to [Fe–S] cluster oxidation and loss in air. Here we highlight tested protocols and strategies enabling efficient and stable [Fe–S] protein production, purification, crystallization, X-ray diffraction data collection, and structure determination. From multiple high-resolution anaerobic crystal structures, we furthermore analyze exemplary data defining [Fe–S] clusters, substrate entry, and product exit for the functional oxidation states of type II molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) enzymes. Notably, these enzymes perform electron shuttling between quinone pools and specific substrates to catalyze respiratory metabolism. The identified structure–activity relationships for this enzyme class have broad implications germane to perchlorate environments on Earth and Mars extending to an alternative mechanism underlying metabolic origins for the evolution of the oxygen atmosphere. Integrated structural analyses of type II Mo-bisMGD enzymes unveil novel distinctive shared molecular mechanisms for dynamic control of substrate entry and product release gated by hydrophobic residues. Collective findings support a prototypic model for type II Mo-bisMGD enzymes including insights for a fundamental molecular mechanistic understanding of selectivity and regulation by a conformationally gated channel with general implications for [Fe–S] cluster respiratory enzymes.

Introduction

Life evolved in an anaerobic world, which can frequently be overlooked from the focus of most funded research on aerobic eukaryotes. Due to this anaerobic origin, our experimental approaches must consider fundamental enzymatic mechanisms and biochemical pathways that were developed and integrated into metabolism in the absence of selective pressure to avoid oxygen reactivity (Imlay, 2008). The [Fe–S] cluster is prone to oxygen sensitivity due to its redox properties enabling conversion from Fe^2 + to Fe^3 +, and the fact that its redox potential can range from − 700 to + 450 mV (Bak & Elliott, 2014; Imlay, 2006). As the redox potential of molecular oxygen ranges from + 300 to + 800 mV (Segel, 1976), the aerobic purification of [Fe–S] proteins can make [Fe–S] clusters prone to oxidation resulting in the loss of [Fe–S] clusters and consequently protein dysfunction and even domain misfolding, as seen for XPD (Fan et al., 2008). Such oxidative damage has biological and practical relevance as oxidation causing mis-metalation can occur in cells, especially under conditions of oxidative stress (Cotruvo & Stubbe, 2012; Imlay, 2014). In aerobic cells, reactive oxygen species are scavenged by oxygen into superoxide that can accelerate DNA and other damage by destroying [Fe–S] clusters with a rate constant of 10⁶–10⁷ M^− 1 s^− 1 and elevating free-iron levels (Imlay, 2008; Keyer & Imlay, 1996). As a result, superoxide dismutase metalloenzymes, which convert superoxide radicals to molecular oxygen and hydrogen peroxide that is further reduced to oxygen by peroxidases and catalases, are critical to the stability of [Fe–S] cluster enzymes in many cells including many anaerobes that may generate oxygen during metabolism (Imlay, 2008; Perry, Shin, Getzoff, & Tainer, 2010). The fundamental sensitivity of [Fe–S] clusters to superoxide and nitric oxide, which is tightly controlled by its synthesis from arginine in eukaryotes (Crane et al., 1998) and some microbes (Adak et al., 2002), drove the development of multiple protective superoxide dismutases with several different folds and metal ion active sites (Perry et al., 2010). In human cells, DNA sequence regions without stable double-helical conformations are especially vulnerable to iron-mediated damage, and translocation breakpoints in patient tumor samples are associated with these regions (Bacolla, Tainer, Vasquez, & Cooper, 2016). Strikingly, in higher eukaryotes both growth defects and DNA damage caused by superoxide develop primarily from its toxic damage to iron–sulfur clusters (ISCs) (Keyer & Imlay, 1996).

Whenever doing experiments with [Fe–S] cluster proteins, it is therefore crucial to appreciate dioxygen toxicity to [Fe–S] clusters, despite the fact that they are truly ancient cofactors essential for all domains of life (Johnson, Dean, Smith, & Johnson, 2005; Lill, 2009). Cubane [4Fe–4S] and rhombic [2Fe–2S] clusters are those found most frequently in proteins (Beinert, 1997; Johnson et al., 2005) although more complex [Fe–S] clusters such as H-cluster, P-cluster, and FeMo cofactor also exist (Broderick et al., 2014; Burgess & Lowe, 1996; Hoffman, Lukoyanov, Yang, Dean, & Seefeldt, 2014). They play essential roles for protein function and are even required for DNA replication, transcription, and repair critical for genome integrity and stability (Fuss, Tsai, Ishida, & Tainer, 2015) where their function may include detection of non-B-DNA areas, which are generally associated with increased breaks and translocations linked with cancer (Bacolla et al., 2016). In prokaryotes, the formation, regulation, and diversity of [Fe–S] clusters have been most studied for Escherichia coli and Azotobacter vinelandii (Frazzon & Dean, 2003; Roche et al., 2013). In eukaryotes, maturation of both cytosolic and nuclear Fe–S proteins requires the cooperation of the mitochondrial systems derived from microbial ancestors (Lill & Mühlenhoff, 2006; Paul & Lill, 2015). In fact, [Fe–S] cluster proteins are key to mitochondrial respiration including the assembly, stability, and function of respiratory complexes I, II, and III, which underscores the fundamental roles of Fe–S clusters in biological respiration (Melber et al., 2016). The maturation and insertion of [Fe–S] clusters into apo-target proteins require mitochondrial ISC assembly machinery that contains scaffold protein IscU, cysteine desulfurase (Nfs1), accessory protein Isd11, activator/iron donor Frataxin, and Ferredoxin (Agar et al., 2000; Colin et al., 2013; Cory et al., 2017; Johnson et al., 2005; Lill, 2009; Pandey et al., 2013; Raulfs, O'Carroll, Santos Dos, Unciuleac, & Dean, 2008; Schmucker et al., 2011; Tsai & Barondeau, 2010; Webert et al., 2014; Yoon & Cowan, 2003; Zheng, Cash, Flint, & Dean, 1998). [Fe–S] clusters are assembled in mitochondrial and cytosolic machineries (Braymer & Lill, 2017; Bridwell-Rabb, Fox, Tsai, Winn, & Barondeau, 2014; Lill et al., 2006), and clusters are transferred to apo-target proteins aided by chaperones HscA/HscB upon maturation (Bonomi, Iametti, Morleo, Ta, & Vickery, 2011; Chandramouli & Johnson, 2006; Fox, Chakrabarti, McCormick, Lindahl, & Barondeau, 2015; Fox, Das, Chakrabarti, Lindahl, & Barondeau, 2015; Uzarska, Dutkiewicz, Freibert, Lill, & Mühlenhoff, 2013; Vranish et al., 2015). Defects in Fe–S cluster assembly and maturation lead to multiple human diseases and protein dysfunctions (Rouault, 2015; Stehling, Wilbrecht, & Lill, 2014) that encompass many processes including metabolism, DNA maintenance, Fe–S cluster assembly machineries, respiratory chain metabolism, ribosome function, and tRNA modification (Andreini, Banci, & Rosato, 2016). The roles of [Fe–S] clusters include key functions in gene expression activation, substrate binding, electron transfer, sulfur donation, and structural stabilization when ligated to protein cysteine residues (Johnson et al., 2005). Of these many functions, a remarkable and exemplary function in [Fe–S] proteins is electron shuttling, which was proposed as the rate-limiting step, between multi-[Fe–S] clusters functioning in respiratory metabolism (de Vries, Dörner, Strampraad, & Friedrich, 2015; Martin & Matyushov, 2017; Rikken, Kroon, & van Ginkel, 1996).

For such respiratory electron transfer, an underappreciated and critical [Fe–S] function involves the respiration of perchlorate (${{\mathrm{ClO}}_4}^{-}$) and chlorate (${{\mathrm{ClO}}_3}^{-}$) [collectively, (per)chlorate] that occurs in dissimilatory (per)chlorate-reducing bacteria (Youngblut, Wang, Barnum,& Coates, 2016). Perchlorate is a water-soluble chemical that can be reduced to chlorate and subsequently reduced to chlorite (${{\mathrm{ClO}}_2}^{-}$) by (per)chlorate reductase (PcrAB). The product chlorite, a harmful molecule, is then dismutated to chloride and oxygen by chlorite dismutase (Cld) (Lee, Streit, Zdilla, Abu-Omar, & DuBois, 2008) in a manner somewhat reminiscent of the superoxide dismutase reaction (Perry et al., 2010). The oxygen is further used by cytochrome cbb₃ oxidase for quinone oxidation (Bak & Elliott, 2014; Buschmann et al., 2010; Coates & Achenbach, 2004; Hosler, Ferguson-Miller, & Mills, 2006; Imlay, 2006). A remarkable feature of (per)chlorate respiration is electron shuttling (2e⁻ at a time) from the quinone pool to heme to five [Fe–S] clusters, and finally to the Mo-bisMGD cofactor for (per)chlorate reduction (Youngblut, Tsai, et al., 2016; Youngblut, Wang, et al., 2016). The structure–activity relationships for these enzymes are relevant to removal of perchlorate (${{\mathrm{ClO}}_4}^{-}$) and nitrate (${{\mathrm{NO}}_3}^{-}$) from drinking water (Matos, Velizarov, Crespo, & Reis, 2006), to environmental controls of perchlorate and nitrate co-occurrence in arid and semiarid environments (Jackson, Böhlke, et al., 2015), to the control of H₂S production from industrial oil recovery and mining (Carlson et al., 2015), and even to the generation of water and oxygen from perchlorate on Mars (Carlson et al., 2015; Ojha et al., 2015). Given its emblematic and understudied features, we focus on PcrAB to examine relationships of protein structure to the regulation of [Fe–S] cluster function.

Importantly, examination of PcrAB enzyme structures discovered distinct conformational states in reduced and oxidized [Fe–S] cluster forms that inform a detailed mechanism for (per)chlorate reduction. These structures showcase the fundamental importance of [Fe–S] cluster enzymes for cellular energy and their dramatic effects on altering protein functional conformations. The identified aromatic gate residues guard substrate entry and regulate product release to control the electron flow for (per)chlorate reduction (Youngblut, Tsai, et al., 2016). Yet, we find that in light of comparative structural analyses, the seemingly unique aromatic gate opening and closing for PcrAB may in fact serve as a prototypic model for Mo-bisMGD- and [Fe–S] cluster-containing respiratory proteins, which can be usefully grouped into the DMSO reductase family (Schindelin, Kisker, Hilton, Rajagopalan, & Rees, 1996; Youngblut, Tsai, et al., 2016). To furthermore robustly enable research on [Fe–S] cluster-containing proteins, we herein share our tactics, protocols, and strategies for [Fe–S] protein production, purification, crystallization, data collection, and X-ray structure determination and use PcrAB as a prototypic system for structure analysis.