The analysis of inquiry in students' conversations in the biochemistry laboratory : the elucidation of proton-coupled electron-transfer reaction mechanism in manganese superoxide dismutase through structural analysis of mutants : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand
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Date
2023
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Massey University
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Abstract
Superoxide dismutases (SODs) have very significant biological importance, protecting organisms against reactive oxygen species such as superoxide. They are also known as the fastest enzyme with the largest kcat/Km of any known enzyme. To perform super-fast enzymatic function, SOD must shuttle proton-coupled electrons in an efficient systematic way. However, since its discovery in 1968, the mechanistic nature of SOD catalytic function remains vague. Wide-ranging approaches have attempted to uncover the catalytic mechanism of the manganese-containing SOD, MnSOD, but there were experimental limitations that obstructed the investigations. Here, the structural analyses of two dimer interface mutants of MnSOD, S126D and S126W, explored possible changes in water structure near the active site providing new information to examine the hypothesis of the Glu170 bridge as a key player in the proton shuttle in the outer-sphere mechanism.
To gain insight into the mechanism of the proton-coupled electron-transfer (PCET) reaction mechanism, the technique of single-crystal X-ray crystallography was used to observe the three-dimensional structure of Escherichia coli MnSOD mutants, analytical ultracentrifugation was used to observe quaternary association in solution, and protein stability was assessed by differential scanning calorimetry. The key residue Ser126 at the conserved but asymmetric dimer interface of the MnSOD was mutated with the initial intent to generate a monomeric species. Ser126 is not essential for activity and is not part of the active site, whereas Glu170 forms part of the dimer interface where Glu170 from one subunit forms part of the active site of the second subunit of the dimer. The loss of activity occurring in a monomeric MnSOD may indicate an alternative catalytic mechanism of the MnSOD enzyme.
The substitution of Ser126 to Asp, intended to produce a monomeric species by charge repulsion, surprisingly produced a dimer at pH>7.5 with little change in structure at the Mn active site, but there was a 94 % reduction in catalytic activity. Partial loss of activity in Ec-MnSOD-S126D may be due to electrostatic effects of the negative charge ~7 Å from metal centre perturbing the Mnᴵᴵᴵ/Mnᴵᴵ redox couple.
The substitution of Ser126 to Trp, intended to produce a monomeric species by steric bulk, enforces mostly monomeric Ec-MnSOD S126W in solution form, coupled with a 99.9 % reduction in catalytic activity. Here one mutation to a conserved dimer interface led to altered tertiary structure and a completely different dodecameric domain-swapped quaternary association in the crystalline state and complete loss of activity in Ec-MnSOD-S126W in the solution state. In the course of evolution, higher and less often lower degrees of oligomerisation have arisen. Evolving complexity does not require multiple mutations.
As part of the scholarship requirements, this dissertation contains a pedagogical component. Student conversations in a guided inquiry third-year biochemistry laboratory were recorded and analysed to discover the extent of higher-order critical thinking that might occur. Although students initially struggled to move beyond core first-year laboratory skills, they were at all times strongly engaged in the project-style experiment, which ran over three five- to eight-hour sessions. Some progress in the level of inquiry was captured from their conversations from the first to the third laboratory session. A simple diagram and table were developed to help guide teachers in a guided inquiry-based learning in higher education.
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Biochemistry, Study and teaching (Higher), College students, Research, Oxidation-reduction reaction, Oxidoreductases, Enzymes, manganese superoxide dismutase, MnSOD, dimer interface mutants, proton-coupled electron-transfer, X-ray diffraction, synchrotron, domain swapping, inquiry based learning, student conversation, biochemistry laboratory, teaching laboratory