Persistence of cytochrome c binding to membranes at physiological mitochondrial intermembrane space ionic strength

Abstract

We have shown that cytochrome c (cyt c) diffuses primarily in three dimensions in the intermembrane space (IMS) of intact mitochondria at physiological ionic strength (I). Recently, we found that a small percentage (11.2 +/- 2.1%) of endogenous cyt c remains bound to inner mitochondrial membranes (IMM) at high, physiological I (I = 150 mM), even after extensive washing with solutions at physiological I, overnight dialysis, changes in medium osmolarity, or further purification of IMM at high I using self-generating Percoll gradients. Measurements of heme c/heme a ratios, and electron transport (ET) reactions in which cyt c participates, confirmed the presence of a low amount of this I-resistant, membrane-bound form of cyt c (MB-cyt c), that had one third of the ET activity of electrostatically-bound cyt c (EB-cyt c), and which could not account for maximal ET rates. The amount of MB-cyt c was significantly increased above endogenous MB-cyt c by exposing KCl-washed IMM to increasing concentrations of exogenous cyt c. Also, subjecting large unilamellar vesicles (LUV) to successive cycles of cyt c binding/high I KCl-washes gave progressive increases in MB-cyt c. These protocols allowed in vitro characterization of MB-cyt c. The I at which binding takes place affects the affinity of cyt c for membranes, and oxidized cyt c had a greater intrinsic affinity for IMM or SUV than reduced cyt c. MB-cyt c appears to be bound partially by hydrophobic interactions since MB-cyt c was detected on negatively charged (asolectin) LUV and also on neutral, zwitterionic (phosphatidylcholine) LUV at high I. Consistent with the concentration-dependent changes in MB-cyt c, decreasing the IMS-volume of intact mitochondria (i.e., increasing th endogenous IMS-cyt c concentration) by metabolic or osmotic means increased the amount of MB-cyt c. After cyt c was delivered into the IMS by liposome-mediated low pH-induced fusion, resonance energy transfer showed a time-dependent cyt c-membrane proximity which was consistent with slow exchange of soluble IMS-entrapped cyt c molecules with a population bound to membranes at I = 150 mM. We conclude that, even though the majority of functional IMS-cyt c diffuses in three dimensions, a small portion remains firmly bound on the surface of the IMM under I conditions that are physiological for intact mitochondria. The occurrence of MB-cyt c may reflect an intrinsic conformational flexibility in cyt c, that allows a degree of membrane penetration and the formation of hydrophobic interactions which stabilize the membrane-bound form. The persistence of cyt c-membrane interactions under physiological I conditions indicates that cyt c-mediated ET in the IMS involves both fast (3D-diffusion) and slow (2D-diffusion) pathways for electron transfer.

MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity

“…Cytochrome c, which associates with the inner membrane, was stripped efficiently from the mitochondria under these conditions (Fig. 4A) as expected (17,18). In contrast, mitoNEET remained associated with the mitochondrial pellet as did other known integral membrane proteins such as BCL2 and voltage-dependent anion channel protein (Fig.…”

Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells

“…Cytochrome c normally resides in the inter-membrane space of the mitochondria and is involved in energy transfer (38). However, a population of cytochrome c is released into the cytosol during apoptosis.…”

Mitochondrial Membrane Permeabilization and Superoxide Production during Apoptosis

“…Cyt-c may function as a direct antioxidant in living cells (26). It is known that a large fraction of cyt-c does not participate in electron transport (27) and hence that cyt-c is present in excess in the mitochondrial intermembrane space. Electron microscopy studies have indicated that the release of cyt-c during apoptosis is a complete process, leading to a total depletion of cyt-c in affected mitochondria (28).…”