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

Abstract

Molecular motion of metal-free and metal-substituted 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. We find that the anisotropy of fluorescence of metal-free cytochrome c when bound to 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 zinc cytochrome c, as measured by triplet-triplet absorption and phosphorescence emission is z 9 ms for free zinc cytochrome c and 7 ms for mitochondrial membrane-bound zinc cytochrome c at room temperature. The decay of anisotropy of phosphorescence emission of mitochondrial membrane-bound zinc cytochrome c is clearly biphasic; the fast component corresponds to a rotational relaxation time of 300 ps and the slow component with relaxation time of z 6 ms.The slow component appears 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.The question of organization of the mitochondrial electron transfer components is fundamental to the understanding of electron transfer and ATP coupling. There are several lines of evidence that the high rates and specificity of electron transfer in mitochondria is achieved through precise structural organization in terms of location and orientation of the components of the mitochondrial respiratory chain. For example, the hemes and iron-sulfur centers appear to be oriented with respect to the plane of the membrane [I -31.On the other hand, the possibility of lateral diffusion of the membrane proteins is an important aspect in understanding the electron transport mechanism. The electron transfer in the respiratory chain of mitochondria between the reductase and the oxidase, and the role of cytochrome c in the process presents an interesting question concerning the motion and the organization of the integral proteins. Cytochrome c is a small peripheral membrane protein which provides a path for electron transfer between two large intrinsic membrane proteins, the cytochrome bcl complex and the cytochrome oxidase. Because of this relationship, an intriguing question is raised: what type and what degree of molecular motion are required for the interaction of cytochrome c with both the oxidase and the reductase? There are several reports in the literature indirectly implying the motion of integral proteins [4-61. In this paper we discuss several types of molecular motion (whole molecule and molecular structural fluctuation) in metal-free and metal-substituted cytochrome c, utilizing the anisotropy of emissi...

Role of mobility of redox components in the inner mitochondrial membrane

“…The kinetics of electron transfer from cyt.c through the oxidase to molecular oxygen exhibit two or three phases (Thompson, Suarez & Ferguson-Miller, 1982); the polarographically determined rate of 02 reduction is much faster than the net rate of spectroscopic appearance of oxidized cyt.c (Ferguson-Miller, Brautigan & Margoliash, 1978), suggesting that the initial rapid phase is due to multiple turnovers of bound cyt.c prior to dissociation from the oxidase. Consistently, a phosphorescent cyt.c derivative rotates at the same slow rate as cytochrome oxidase bound to mitochondrial membranes (Dixit et al, 1982), suggesting complexation, and cyt.c covalently bound to either the reductase or the oxidase is still capable of mediating electron transfer (Erecinska, Davis & Wilson, 1980;Waring et al, 1980); a mechanism of rapid oscillation of cyt.c between reductase and oxidase could explain fast respiratory rates. Hackenbrock et al (1986) severely criticized the above interpretation on the basis that under physiological conditions of 150 mM ionic strength, cytochrome c is readily dissociated from the membrane and appears free to undergo three-dimensional diffusion (Gupte et al, 1984) in the intermembrane space; accordingly, duroquinol oxidase activity is enhanced by increasing ionic strength in parallel with the diffusion coefficient of cyt.c.…”

“…No immobile fraction of cyt.c was found at high ionic strength by Gupte et al (1984), whereas Vanderkooi et al (1985 found that a fraction of 3 nmol of cyt.c derivative per mg protein remained immobile and tightly bound to the membrane. Dixit et al (1982) found that a phosphorescent cyt.c derivative rotates at the same slow rate as cytochrome oxidase bound to mitochondrial membranes, suggesting complexation between the two molecules; the smaller radius of cyt.c, in fact, would make its rotation much faster if the molecule were completely free.…”

Mobility of fluorescent derivatives of cytochrome c in mitochondria.

“…Consistent with this idea is the finding that only about half of the cytochrome c oxidase appears to be rotationally mobile (17,18). The rotational mobility of zinc cytochrome c also showed biphasicity which was interpreted to be due to rotation in a cone, but could also be due to an immobilized fraction (6). Some mobility of the electron-transfer components is, however, suggested by observations indicating the independent diffusion of cytochrome oxidase and cytochrome bcl complex (13) and their electrophoretic mobility (25) as well as by electron micrographs of inner mitochondrial membranes labeled with antibodies against oxidase and reductase which show random distribution (12).…”

Cytochrome c mediates electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase by free diffusion along the surface of the membrane

“…As an alternative explanation for the results of Hochman et al (1982), it may be that there is some constraint to long-range lateral diffusion (as measured by fluorescence recovery after photobleaching) and that only short-range motions are necessary in the mitochondrial membrane (Cherry, 1979;Kawato et al, 1981). Dixit et al (1982) measured the rotational diffusion of zinc cytochrome c on mitochondrial membranes by using the decay of phosphorescence anisotropy. The rotational relaxation time was similar to that measured for cytochrome oxidase (Kawato et al, 1981), suggesting that cytochrome c is associated with its oxidase in situ.…”