Serial Review: The powerhouse takes control of the cell: The role of mitochondria in signal transduction Serial Review Editor: Victor Darley-Usmar
Mitochondrial H+ leak and ROS generation: An odd couple

https://doi.org/10.1016/j.freeradbiomed.2004.10.016Get rights and content

Abstract

The single-electron chemistry of mitochondrial oxidative phosphorylation (ox-phos) by default generates reactive oxygen species (ROS). These ROS have roles in both physiologic cell signaling and numerous pathologic situations. One factor that has the potential to regulate ROS generation is the mild uncoupling of ox-phos, i.e., proton (H+) leak across the mitochondrial inner membrane. Proton leak has been shown to decrease ROS generation, whereas ROS have been shown to induce H+ leak, and this suggests the existence of a feedback loop between ROS and H+ leak. Interestingly, although H+ leak is detrimental to ATP synthesis, it has been shown to be cytoprotective in several models of ischemic injury. Herein the molecular basis of both ROS generation and H+ leak will be reviewed and the consequences of their interaction for mitochondrial function discussed.

Section snippets

Mitochondrial oxidative phosphorylation

Whereas a full review of the nature of mitochondrial oxidative phosphorylation (ox-phos) is beyond the scope of this review, the basic layout of the respiratory chain and the chemiosmotic principle of energy conservation are shown in Fig. 1. Protons are pumped out across the mitochondrial inner membrane by complexes I and IV and by the Q cycle at complexes I, II, and III. The energy of the resulting trans-membrane H+ electrochemical potential gradient (the proton-motive force, ΔμH+) is used by

Mitochondrial ROS generation

Mitochondria are a significant cellular source of reactive oxygen species (ROS) and under nonpathological conditions may indeed be the dominant cellular source of ROS [2], [3], [4], [5], [6], [7]. The primary ROS generated in the organelle is superoxide (O2radical dot), which is then converted to H2O2 by spontaneous dismutation or by superoxide dismutase (SOD). The main mitochondrial O2radical dot source was until recently believed to be the ubisemiquinone radical (QHradical dot), formed at the complex III QO site facing the

Mitochondrial H+ leak/uncoupling

The integrity of the inner mitochondrial membrane (IMM) is essential to the function of ox-phos. However, it has been known for several decades that the IMM is partially permeable to protons [17]. This H+ leak is termed “non-ohmic” because it is related to the driving force (Δψm) in a nonlinear fashion (i.e., disobeying Ohm's law) and increases exponentially at high values of Δψm. The H+ leak as been characterized in isolated mitochondria [18], and also in mitochondria inside intact cells [19]

The basal H+ leak

In quantifying H+ leak it is important to delineate between the basal leak and that which is inducible by a variety of physiologic and pathologic conditions and mediated by specific leak proteins (e.g., the uncoupling proteins, UCPs). The basal leak is significant, as evidenced by the H+ leak of mitochondria from mice with their UCPs genetically ablated. However, efforts to determine the molecular mechanism of the basal H+ leak have been largely unsuccessful.

The basal leak is not a mere leakage

Inducible H+ leak—the uncoupling proteins

The existence of regulated H+ leak proteins in the mitochondrial inner membrane continues to be a source of much controversy. The mitochondria of brown adipose tissue (BAT) are known to possess an uncoupling protein (termed “thermogenin” or UCP), which is responsible for heat generation in this organ [34]. In 1997, a series of UCP homologs (UCP2–6, herein termed UCPn) was identified in other tissues [35], [36], [37] and named UCPs on the basis of their sequence similarity (∼50%) to BAT UCP.

The

Inducible H+ leak—the AMP/ANT pathway

The ANT is one of the most abundant proteins of the inner mitochondrial membrane [27], accounting for nearly 10% of all the protein in cardiac mitochondria. It has been shown that adenosine monophosphate can allosterically act on the ANT to induce a H+ leak [56]. This inducible leak is inhibited by the specific ANT inhibitor carboxyattractyloside [56] and is also absent in skeletal muscle mitochondria from ANT-1−/− mice (E.C. Cornwall, P.S. Brookes, M.D. Brand, unpublished data). Further

Effects of H+ leak on ROS generation

Several authors report that dissipation of Δψm by chemical uncouplers or the presence of ADP decreases the rate of H2O2 generation [4], [10], [59]. Although conflicting reports of an uncoupling-mediated increase in ROS generation exist [2], [4], [13], the important overriding factor seems to be the presence of respiratory inhibitors; without becoming mired in the experimental detail of each investigation, this large body of work can be summarized as showing that uncoupling of mitochondria

Effects of ROS on H+ leak

In addition to the effects of H+ leak on ROS, there are also well-documented effects of ROS on H+ leak. In model membrane systems, phospholipid oxidation causes an increase in H+ leak [79], a decrease in electrical resistance [80], and a decrease in membrane thickness [81]. In addition we have observed that peroxynitrite (ONOO; the product of the reaction between O2radical dot and NOradical dot and a potent inducer of lipid oxidation) caused an increase in the H+ leak of isolated brain mitochondria [82]. This

The ROS/H+ leak feedback loop

From the experimental evidence discussed above, it seems that mitochondrial H+ leak can decrease O2radical dot generation and that mitochondrially generated O2radical dot can increase H+ leak. Thus, a feedback loop may exist between ROS and H+ leak (Fig. 5) [12], [92]. Several fundamental aspects of this feedback system remain to be determined, most importantly the mechanism(s) by which ROS can stimulate H+ leak. Although a role for UCPs in this feedback loop cannot be excluded, UCPs are not necessary for the loop

Coming full circle: ROS generation is a form of H+ leak

In bioenergetic terms mitochondrial H+ leak is essentially defined as a lower than predicted H+/e stoichiometry. That is, for a given number of electrons flowing down the respiratory chain to O2, a lower number of protons are pumped than expected, and a lower Δψm is generated. Phrased alternatively, the number of electrons flowing down the chain that are required to maintain a given Δψm is higher than expected. In classical bioenergetics, two alternative mechanisms were proposed to explain

Closing remarks

Clearly, the phenomenon of H+ leak has come a long way from the bioenergetic backwater in which it stood 10 years ago. The realization that mitochondrial H+ leak and ROS generation are a mechanistically linked “odd couple” has provided important insight into the field of H+ leak research, with implications for obesity, diabetes, and energy metabolism. In addition H+ leak is making significant headway into the field of mitochondrial ROS research, with implications for cell signaling and

Acknowledgments

This research was funded by the NIH (HL71158). I am indebted to several people for valuable discussions on these topics, including Victor Darley-Usmar, Carlos Krumdieck, and Tim Nagy (Birmingham, AL, USA); Florian Muller (San Antonio, TX, USA); and Martin Brand and Aubrey DeGrey (Cambridge, UK).

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    This article is part of a series of reviews on “The Powerhouse Takes Control of the Cell: The Role of Mitochondria in Signal Transduction.” The full list of papers may be found on the home page of the journal.

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