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

• Published in: Methods in enzymology Vol. 599; p. 157
• Main Authors: Tsai, Chi-Lin, Tainer, John A
• Format: Journal Article
• Language: English
• Published: United States 01.01.2018
• Subjects: Animals; Crystallization - methods; Crystallography, X-Ray - methods; Electrons; Guanine Nucleotides - metabolism; Humans; Iron-Sulfur Proteins - chemistry; Iron-Sulfur Proteins - genetics; Iron-Sulfur Proteins - metabolism; Models, Molecular; Oxidation-Reduction; Protein Conformation; Pterins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Substrate Specificity; Anaerobic purification and crystallization; Substrate tunnel; Respiratory metabolism; Electron shuttling; Mo-bisMGD enzymes; Fe–S protein
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2017.11.006

[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.