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Since deconvolution assumes equilibrium conditions (and thus slow scan rates), the ET rates to the [3Fe-4S] and [2Fe-2S] clusters cannot be measured in this way.
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The reduction potential varies slightly with buffer concentration; see Materials and Methods.
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41
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This equation (described in ref 7 and in: Page, C. C.; Moser, C. C.; Dutton, P. L. Curr. Opin. Chem. Biol. 2003, 7, 551-556) describes the ET rate constant within proteins in terms of the distance between centers, the packing density of the protein, the change in free energy, and the reorganization energy. The calculation included a standard value for the protein packing density as 0.75, reorganization energy of the Fe-S clusters as 0.7 eV, and the edge-edge distances from the WT crystal structure. The ET rate for each mutant was based on the step from the [3Fe-4S] to the [4Fe-4S] cluster since this is the most energetically unfavorable step and would most likely be rate-determining if energetics were to control the inherent kinetics of electron flow along the relay.
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These OAA release rates were measured in the presence of chloride, which (like other anions) has been shown to bind to the active site. The presence of chloride in these experiments is responsible for the higher release rates and weaker binding reported here compared to those measured previously (see ref 13).
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18744364082
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It is known also that malate can be oxidized to OAA by SQR (ref 38), thus enhanced release of OAA by a very slow catalytic reduction process, which might somehow be enhanced by the delivery of two electrons, also remains a possibility. Although OAA reduction could not be detected with PFV, more sensitive gas chromatography analysis of the products after bulk catalysis may provide more evidence for this possibility.
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