Rotational Motion of Cytochrome c Derivatives Bound to Membranes Measured by Fluorescence and Phosphorescence Anisotropy

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Abstract
Molecular motion of metal-free and metal-substituted [horse heart] cytochrome c derivatives was examined using the anisotropy of emissions from the singlet and the triplet states. The anisotropy of fluorescence provides a means to study the motion of cytochrome c in the nanosecond time scale, since the fluorescence lifetime of metal-free cytochrome c is around 10 ns. The anisotropy of fluorescence of metal-free cytochrome c when bound to [rat liver] mitochondria does not decay, but, when bound to phospholipids, has a small component which decays independently of the rotation of the whole molecule. The use of phosphorescence extends the time scale for study into the millisecond regime, since the lifetime of the excited triplet state of Zn cytochrome c as measured by triplet-triplet absorption and phosphorescence emission is .apprxeq. 9 ms for free Zn cytochrome c and 7 ms for mitochondrial membrane-bound Zn cytochrome c at room temperature. The decay of anisotropy of phosphorescence emission of mitochondrial membrane-bound Zn cytochrome c is clearly biphasic; the fast component corresponds to a rotational relaxation time of 300 .mu.s and the slow component with relaxation time of .apprxeq. 6 ms. The slow component appeared to be due to the rotation of the entire mitochondrion, whereas the fast component was interpreted to be due to the rotation of cytochrome c in a cone about a single axis perpendicular to the plane of the membrane surface.

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