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From Structure to Function: Utilizing the Biophysical Toolbox to Interrogate a Novel Class of Mn/Fe Proteins

Miller, Effie Kisgeropoulos
http://orcid.org/0000-0003-1842-5691
http://rave.ohiolink.edu/etdc/view?acc_num=osu1588957858885161

Abstract Details

2020, Doctor of Philosophy, Ohio State University, Biochemistry Program, Ohio State.
The recently discovered Mn/Fe proteins represent a paradigm shift in the field of bioinorganic chemistry. These proteins defy conventional inorganic wisdom, going against the commonly accepted Irving-Williams series of divalent metal binding stability to selectively bind a MnII/FeII metal core. While the purple acid phosphatase protein from sweet potato was the first example of a biologically occurring Mn/Fe protein, its metals are not redox-active during enzyme catalysis. A few years later, two other Mn/Fe proteins were discovered that utilize their metallocofactor to perform either one- or two-electron oxidative chemistry. The first was the R2 subunit of a class Ic (R2c) ribonucleotide reductase (RNR) from Chlamydia trachomatis, whose heterobimetallic active site defined the new, Ic subclass of RNR proteins. Like other RNR proteins, R2c is only capable of performing single-electron, radical chemistry, although it does so by storing its oxidizing equivalent on metal center in the form of a high-valent, MnIV/FeIII cofactor, rather than generating a nearby, stable tyrosine radical. Around the same time, another Mn/Fe protein was discovered that demonstrated the ability to perform oxidative, two-electron chemistry. Initially isolated from Mycobacterium tuberculosis, the R2-like ligand-binding oxidase (R2lox) protein is found within various pathogens and extremophiles and performs selective C-H bond oxidation upon binding and activating oxygen to generate a novel, tyrosine-valine crosslink within its scaffold. This reactivity is similar to that seen in the diiron bacterial multicomponent monooxygenases (BMMs), such as soluble methane monooxygenase (sMMO), and engenders many questions regarding the scope of reactivity available to the Mn/Fe cofactors, as well as the molecular factors that might help tune this reactivity. R2lox also contains a hydrophobic channel that extends down into the active site, reminiscent of the substrate binding channel found in some BMM proteins, and in R2lox a mixture of long-chain fatty acids is found bound in this channel. While the fatty acids present upon heterologous expression do not appear to be functionally relevant, the carboxylate head group of this ligand coordinates directly to the metal center in a bridging fashion, potentially demonstrating a mode of substrate binding. In this work, the Mn/Fe protein R2lox from Geobacillus kaustophilus forms the cornerstone of an investigation into connections between structure, reactivity, and, ultimately, function, in biological systems. Using the biophysical toolbox, these connections are interrogated using a combined approach of various steady-state and time-resolved spectroscopic techniques, kinetic modeling, and molecular biology and protein engineering methods. In the first half of this thesis, the assembly mechanism of the MnIII(µ-OH)FeIII cofactor is examined, with multiple reaction intermediates identified that have distinct kinetic profiles and optical and electronic signatures. Targeted mutation of the R2lox primary and secondary coordination sphere provides additional insight into the molecular factors influencing the initial Mn/Fe cofactor formation, as well as reactivity towards crosslink formation. The second half of this thesis focuses specifically on the use of electron paramagnetic resonance (EPR) spectroscopy as an ideal tool to investigate structure/reactivity relationships in biological systems. Both continuous-wave (CW) and pulsed EPR are used to investigate a trapped intermediate of the Mn/Fe R2lox activation process, informing upon the electronic and geometric structure of this novel Mn/Fe species with a potential role in substrate binding in R2lox. These techniques are then applied to characterizing organometallic, Ni-C bond formation that happens during the reaction of a Ni-substituted azurin (NiAz) with either carbon monoxide (CO) or the methyl donor, methyl iodide (CH3I). This NiAz system was developed in the Shafaat group as a model for the acetyl-CoA synthase (ACS) protein, and characterization of these species holds implications for better understanding substrate binding and reactivity in ACS.
Hannah Shafaat (Advisor)
Jane Jackman (Committee Member)
James Cowan (Committee Member)
Amanda Bird (Committee Member)
270 p.
Chemistry
metalloprotein; spectroscopy; R2lox

Recommended Citations

Citations

  • Miller, E. K. (2020). From Structure to Function: Utilizing the Biophysical Toolbox to Interrogate a Novel Class of Mn/Fe Proteins [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1588957858885161

    APA Style (7th edition)

  • Miller, Effie. From Structure to Function: Utilizing the Biophysical Toolbox to Interrogate a Novel Class of Mn/Fe Proteins. 2020. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1588957858885161.

    MLA Style (8th edition)

  • Miller, Effie. "From Structure to Function: Utilizing the Biophysical Toolbox to Interrogate a Novel Class of Mn/Fe Proteins." Doctoral dissertation, Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1588957858885161

    Chicago Manual of Style (17th edition)

osu1588957858885161
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This open access ETD is published by The Ohio State University and OhioLINK.
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