Mobility in the mitochondrial electron transport chain

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.     

Relationship between mitochondrial electron transport chain dysfunction, development, and life extension in Caenorhabditis elegans

Prior studies have shown that disruption of mitochondrial electron transport chain (ETC) function in the nematode Caenorhabditis elegans can result in life extension. Counter to these findings, many mutations that disrupt ETC function in humans are known to be pathologically life-shortening. In this study, we have undertaken the first formal investigation of the role of partial mitochondrial ETC inhibition and its contribution to the life-extension phenotype of C. elegans. We have developed a novel RNA interference (RNAi) dilution strategy to incrementally reduce the expression level of five genes encoding mitochondrial proteins in C. elegans: atp-3, nuo-2, isp-1, cco-1, and frataxin (frh-1). We observed that each RNAi treatment led to marked alterations in multiple ETC components. Using this dilution technique, we observed a consistent, three-phase lifespan response to increasingly greater inhibition by RNAi: at low levels of inhibition, there was no response, then as inhibition increased, lifespan responded by monotonically lengthening. Finally, at the highest levels of RNAi inhibition, lifespan began to shorten. Indirect measurements of whole-animal oxidative stress showed no correlation with life extension. Instead, larval development, fertility, and adult size all became coordinately affected at the same point at which lifespan began to increase. We show that a specific signal, initiated during the L3/L4 larval stage of development, is sufficient for initiating mitochondrial dysfunction–dependent life extension in C. elegans. This stage of development is characterized by the last somatic cell divisions normally undertaken by C. elegans and also by massive mitochondrial DNA expansion. The coordinate effects of mitochondrial dysfunction on several cell cycle–dependent phenotypes, coupled with recent findings directly linking cell cycle progression with mitochondrial activity in C. elegans, lead us to propose that cell cycle checkpoint control plays a key role in specifying longevity of mitochondrial mutants.     

Oxygen tolerance and coupling of mitochondrial electron transport

Oxygen is critical to aerobic metabolism, but excessive oxygen (hyperoxia) causes cell injury and death. An oxygen-tolerant strain of HeLa cells, which proliferates even under 80% O2, termed "HeLa-80," was derived from wild-type HeLa cells ("HeLa-20") by selection for resistance to stepwise increases of oxygen partial pressure. Surprisingly, antioxidant defenses and susceptibility to oxidant-mediated killing do not differ between these two strains of HeLa cells. However, under both 20 and 80% O2, intracellular reactive oxygen species (ROS) production is significantly (approximately 2-fold) less in HeLa-80 cells. In both cell lines the source of ROS is evidently mitochondrial. Although HeLa-80 cells consume oxygen at the same rate as HeLa-20 cells, they consume less glucose and produce less lactic acid. Most importantly, the oxygen-tolerant HeLa-80 cells have significantly higher cytochrome c oxidase activity (approximately 2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. Indeed, preferential inhibition of cytochrome c oxidase by treatment with n-methyl protoporphyrin (which selectively diminishes synthesis of heme a in cytochrome c oxidase) enhances ROS production and abrogates the oxygen tolerance of the HeLa-80 cells. Thus, it appears that the remarkable oxygen tolerance of these cells derives from tighter coupling of the electron transport chain.     

The role of mitochondrial electron transport in tumorigenesis and metastasis

Background

Tumor formation and spread via the circulatory and lymphatic drainage systems is associated with metabolic reprogramming that often includes increased glycolytic metabolism relative to mitochondrial energy production. However, cells within a tumor are not identical due to genetic change, clonal evolution and layers of epigenetic reprogramming. In addition, cell hierarchy impinges on metabolic status while tumor cell phenotype and metabolic status will be influenced by the local microenvironment including stromal cells, developing blood and lymphatic vessels and innate and adaptive immune cells. Mitochondrial mutations and changes in mitochondrial electron transport contribute to metabolic remodeling in cancer in ways that are poorly understood.

Scope of Review

This review concerns the role of mitochondria, mitochondrial mutations and mitochondrial electron transport function in tumorigenesis and metastasis.

Major Conclusions

It is concluded that mitochondrial electron transport is required for tumor initiation, growth and metastasis. Nevertheless, defects in mitochondrial electron transport that compromise mitochondrial energy metabolism can contribute to tumor formation and spread. These apparently contradictory phenomena can be reconciled by cells in individual tumors in a particular environment adapting dynamically to optimally balance mitochondrial genome changes and bioenergetic status.

General Significance

Tumors are complex evolving biological systems characterized by genetic and adaptive epigenetic changes. Understanding the complexity of these changes in terms of bioenergetics and metabolic changes will permit the development of better combination anticancer therapies. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

The role of Coenzyme Q in mitochondrial electron transport

In mitochondria, most Coenzyme Q is free in the lipid bilayer; the question as to whether tightly bound, non-exchangeable Coenzyme Q molecules exist in mitochondrial complexes is still an open question.We review the mechanism of inter-complex electron transfer mediated by ubiquinone and discuss the kinetic consequences of the supramolecular organization of the respiratory complexes (randomly dispersed vs. super-complexes) in terms of Coenzyme Q pool behavior vs. metabolic channeling, respectively, both in physiological and in some pathological conditions. As an example of intra-complex electron transfer, we discuss in particular Complex I, a topic that is still under active investigation.     

Biphasic modulation of the mitochondrial electron transport chain in myocardial ischemia and reperfusion

Mitochondrial electron transport chain (ETC) is the major source of reactive oxygen species during myocardial ischemia-reperfusion (I/R) injury. Ischemic defect and reperfusion-induced injury to ETC are critical in the disease pathogenesis of postischemic heart. The properties of ETC were investigated in an isolated heart model of global I/R. Rat hearts were subjected to ischemia for 30 min followed by reperfusion for 1 h. Studies of mitochondrial function indicated a biphasic modulation of electron transfer activity (ETA) and ETC protein expression during I/R. Analysis of ETAs in the isolated mitochondria indicated that complexes I, II, III, and IV activities were diminished after 30 min of ischemia but increased upon restoration of flow. Immunoblotting analysis and ultrastructural analysis with transmission electron microscopy further revealed marked downregulation of ETC in the ischemic heart and then upregulation of ETC upon reperfusion. No significant difference in the mRNA expression level of ETC was detected between ischemic and postischemic hearts. However, reperfusion-induced ETC biosynthesis in myocardium can be inhibited by cycloheximide, indicating the involvement of translational control. Immunoblotting analysis of tissue homogenates revealed a similar profile in peroxisome proliferator-activated receptor-γ coactivator-1α expression, suggesting its essential role as an upstream regulator in controlling ETC biosynthesis during I/R. Significant impairment caused by ischemic and postischemic injury was observed in the complexes I- III. Analysis of NADH ferricyanide reductase activity indicated that injury of flavoprotein subcomplex accounts for 50% decline of intact complex I activity from ischemic heart. Taken together, our findings provide a new insight into the molecular mechanism of I/R-induced mitochondrial dysfunction.     

Novel interactions between mitochondrial superoxide dismutases and the electron transport chain

The processes that control aging remain poorly understood. We have exploited mutants in the nematode, Caenorhabditis elegans, that compromise mitochondrial function and scavenging of reactive oxygen species (ROS) to understand their relation to lifespan. We discovered unanticipated roles and interactions of the mitochondrial superoxide dismutases (mtSODs): SOD‐2 and SOD‐3. Both SODs localize to mitochondrial supercomplex I:III:IV. Loss of SOD‐2 specifically (i) decreases the activities of complexes I and II, complexes III and IV remain normal; (ii) increases the lifespan of animals with a complex I defect, but not the lifespan of animals with a complex II defect, and kills an animal with a complex III defect; (iii) induces a presumed pro‐inflammatory response. Knockdown of a molecule that may be a pro‐inflammatory mediator very markedly extends lifespan and health of certain mitochondrial mutants. The relationship between the electron transport chain, ROS, and lifespan is complex, and defects in mitochondrial function have specific interactions with ROS scavenging mechanisms. We conclude that mtSODs are embedded within the supercomplex I:III:IV and stabilize or locally protect it from reactive oxygen species (ROS) damage. The results call for a change in the usual paradigm for the interaction of electron transport chain function, ROS release, scavenging, and compensatory responses.     

Heat inactivation of electron transport reactions in photosystem II particles

Heat inactivation of diphenylcarbazide (DPC)-supported 2,6-dichloroindophenol (DCIP) photoreduction by photosystem II (PS II) particles and non-oxygen-evolving PS II core complex isolated from spinach ( Spinacia oleracea L. cv. Kyoho) was suppressed under annealing conditions, and accelerated in the presence of EDTA or high concentration of divalent cations. After heating at 45°C for 10 min, half-maximal annealing effects occurred at 35°C. Minimum acceleration was observed in the presence of 1 m M Mg²⁺, implying the existence of a cation-specific site in the vicinity of the PS II reaction center. The acceleration depended on the temperature at which EDTA was added to PS II particles. Half-acceleration by EDTA occurred at 35°C. Glutaraldehyde stabilized PS II particles against heat inactivation of PS II photochemical reactions.     

The role of cytochrome c diffusion in mitochondrial electron transport

We have compared the modes and rates of cytochrome c diffusion to the rates of cytochrome c-mediated electron transport in isolated inner membranes and in whole intact mitochondria. For inner membranes, an increasing ionic strength results in an increasing rate of cytochrome c diffusion, a decreasing concentration (affinity) of cytochrome c near the membrane surface as well as near its redox partners, and an increasing rate of electron transport. For intact mitochondria, an increasing ionic strength results in a parallel, increasing rate of cytochrome c-mediated electron transport. In both inner membranes and intact mitochondria the rate of cytochrome c-mediated electron transport is highest at physiological ionic strength (100-150 mM), where the diffusion rate of cytochrome c is highest and its diffusion mode is three-dimensional. In intact mitochondria, succinate and duroquinol-driven reduction of endogenous cytochrome c is greater than 95% at all ionic strengths, indicating that cytochrome c functions as a common pool irrespective of its diffusion mode. Using a new treatment to obtain bimolecular diffusion-controlled collision frequencies in a heterogenous diffusion system, where cytochrome c diffuses laterally, pseudo-laterally, or three-dimensionally while its redox partners diffuse laterally, we determined a high degree of collision efficiency (turnover/collisions) for cytochrome c with its redox partners for all diffusion modes of cytochrome c. At physiological ionic strength, the rapid diffusion of cytochrome c in three dimensions and its low concentration (affinity) near the surface of the inner membrane mediate the highest rate of electron transport through maximum collision efficiencies. These data reveal that the diffusion rate and concentration of cytochrome c near the surface of the inner membrane are rate-limiting for maximal (uncoupled) electron transport activity, approaching diffusion control.