Lipophilic phosphonium cations were first used to investigate mitochondrial biology by Vladimir Skulachev and colleagues in the late 1960s. Since then, these molecules have become important tools for exploring mitochondrial bioenergetics and free radical biology. Here we review why these molecules are useful in mitochondrial research and outline some of the ways in which they are now being utilized.
The development of efficient and selective luminescent probes for reactive oxygen species, particularly for singlet molecular oxygen, is currently of great importance. In this study, the photochemical behavior of Singlet Oxygen Sensor Green(®) (SOSG), a commercially available fluorescent probe for singlet oxygen, was examined. Despite published claims to the contrary, the data presented herein indicate that SOSG can, in fact, be incorporated into a living mammalian cell. However, for a number of reasons, caution must be exercised when using SOSG. First, it is shown that the immediate product of the reaction between SOSG and singlet oxygen is, itself, an efficient singlet oxygen photosensitizer. Second, SOSG appears to efficiently bind to proteins which, in turn, can influence uptake by a cell as well as behavior in the cell. As such, incorrect use of SOSG can yield misleading data on yields of photosensitized singlet oxygen production, and can also lead to photooxygenation-dependent adverse effects in the system being investigated.
Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondriatargeted derivative of the spin trap ␣-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation ofUCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.
Mitochondria play a central role in redox-linked processes in the cell through mechanisms that are thought to involve modification of specific protein thiols, but this has proved difficult to assess. In particular, specific labeling and quantitation of mitochondrial protein cysteine residues have not been achieved due to the lack of reagents available that can be applied to the intact organelle or cell. To overcome these problems we have used a combination of mitochondrial proteomics and targeted labeling of mitochondrial thiols using a novel compound, (4-iodobutyl)triphenylphosphonium (IBTP). This lipophilic cation is accumulated by mitochondria and yields stable thioether adducts in a thiol-specific reaction. The selective uptake into mitochondria, due to the large membrane potential across the inner membrane, and the high pH of the matrix results in specific labeling of mitochondrial protein thiols by IBTP. Individual mitochondrial proteins that changed thiol redox state following oxidative stress could then be identified by their decreased reaction with IBTP and isolated by two-dimensional electrophoresis. We demonstrate the selectivity of IBTP labeling and use it to show that glutathione oxidation and exposure to an S-nitrosothiol or to peroxynitrite cause extensive redox changes to mitochondrial thiol proteins. In conjunction with blue native gel electrophoresis, we used IBTP labeling to demonstrate that thiols are exposed on the matrix faces of respiratory Complexes I, II, and IV. This novel approach enables measurement of the thiol redox state of individual mitochondrial proteins during oxidative stress and cell death. In addition the methodology has the potential to identify novel redox-dependent modulation of mitochondrial proteins.Changes in the thiol redox state of mitochondrial proteins are significant in a number of cellular processes including the permeability transition, cell death due to calcium loading and oxidative stress, the response of cells to nitric oxide, tumor necrosis factor signaling, commitment to apoptosis, and in regulating respiratory chain function (1-9). However the detailed mechanisms and the proteins involved are uncertain. This is partly because of the technical challenges presented by determining thiol modifications of proteins in general and the difficulties inherent in mitochondrial proteomics. Potential protein thiol alterations include formation of mixed disulfides or internal disulfides from vicinal dithiols, S-nitrosation, and the formation of higher oxidation states (10 -15). The differential reactivity of individual protein thiols and the range of lifetimes of altered redox states can act as signal sensors or transducers to influence mitochondrial function (13-17). Nitric oxide may be a particularly important regulator of mitochondrial protein thiols because it diffuses easily into mitochondria and partitions selectively into the lipid bilayer where it can modify otherwise inaccessible thiols (18 -20). Modification of protein thiols by nitric oxide most likely occurs...
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