Abstract
The hyperfine shifted resonances in the nuclear magnetic resonance (NMR) spectrum of cytochrome c broaden upon addition of cytochrome c peroxidase in a manner which indicates 1:1 reversible complex formation between cytochrome c and cytochrome c peroxidase. Since, in the presence of excess cytochrome c, we see a time-averaged NMR spectrum and not a simple superposition of two distinct spectra from free and complexed cytochrome c, the exchange between the two forms is fast on the NMR time scale, yielding for the off-rate of the complex a lower limit of 200/s. (The on-rate is several order of magnitude greater than the off-rate.) The observed line widths of methyl resonances are, consistent with theory, dependent on the rotational correlation time of the whole macromolecule/macromolecular complex. The widths of the heme ring methyls of cytochrome c in cytochrome c-peroxidase complex are not sensitive to the electronic spin state changes in the cytochrome c peroxidase heme iron on addition of cyanide or fluoride showing that the heme groups of cytochrome c and cytochrome c peroxidase are sufficiently far from each other. The unpaired electron spin density distribution over the heme ring of cytochrome c in cytochrome c-peroxidase complex is slightly altered with respect to free cytochrome c as indicated by observed small changes in the positions of the ring methyl resonances. This could arise from a small conformational change in cytochrome c on complexing with cytochrome c peroxidase.
Published Version
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