Browsing by Author "Kellett, Whitney F."

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    Assigning the EPR Fine Structure Parameters of the Mn(II) Centers in Bacillus subtilis Oxalate Decarboxylase by Site-Directed Mutagenesis and DFT/MM Calculations
    (American Chemical Society, 2014-02-12) Campomanes, Pablo; Kellett, Whitney F.; Easthon, Lindsey M.; Ozarowski, Andrew; Allen, Karen N.; Angerhofer, Alexander; Rothlisberger, Ursula; Richards, Nigel G. J.; Department of Chemistry & Chemical Biology, School of Science
    Oxalate decarboxylase (OxDC) catalyzes the Mn-dependent conversion of the oxalate monoanion into CO2 and formate. EPR-based strategies for investigating the catalytic mechanism of decarboxylation are complicated by the difficulty of assigning the signals associated with the two Mn(II) centers located in the N- and C-terminal cupin domains of the enzyme. We now report a mutational strategy that has established the assignment of EPR fine structure parameters to each of these Mn(II) centers at pH 8.5. These experimental findings are also used to assess the performance of a multistep strategy for calculating the zero-field splitting parameters of protein-bound Mn(II) ions. Despite the known sensitivity of calculated D and E values to the computational approach, we demonstrate that good estimates of these parameters can be obtained using cluster models taken from carefully optimized DFT/MM structures. Overall, our results provide new insights into the strengths and limitations of theoretical methods for understanding electronic properties of protein-bound Mn(II) ions, thereby setting the stage for future EPR studies on the electronic properties of the Mn(II) centers in OxDC and site-specific variants.
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    A Mechanochemical Switch to Control Radical Intermediates
    (American Chemical Society, 2014-06-17) Brunk, Elizabeth; Kellett, Whitney F.; Richards, Nigel G. J.; Rothlisberger, Ursula; Department of Chemistry & Chemical Biology, School of Science
    B12-dependent enzymes employ radical species with exceptional prowess to catalyze some of the most chemically challenging, thermodynamically unfavorable reactions. However, dealing with highly reactive intermediates is an extremely demanding task, requiring sophisticated control strategies to prevent unwanted side reactions. Using hybrid quantum mechanical/molecular mechanical simulations, we follow the full catalytic cycle of an AdoB12-dependent enzyme and present the details of a mechanism that utilizes a highly effective mechanochemical switch. When the switch is “off”, the 5′-deoxyadenosyl radical moiety is stabilized by releasing the internal strain of an enzyme-imposed conformation. Turning the switch “on,” the enzyme environment becomes the driving force to impose a distinct conformation of the 5′-deoxyadenosyl radical to avoid deleterious radical transfer. This mechanochemical switch illustrates the elaborate way in which enzymes attain selectivity of extremely chemically challenging reactions.