David L. Hoffman, Ph.D.

Over the course of evolution, mitochondria have played essential roles in the continued development of higher organisms. Without mitochondria, it is questionable whether multicellular organisms would have evolved at all. Possessing their own DNA and transcription machinery, strong evidence supports that mitochondria were once free-living aerobic bacteria during the Statherian period. Within this period, α-proteobacteria became part of a multicellular system when engulfed by anaerobes. This relationship, described as symbiotic, proved to be mutually beneficial by providing a source of hydrogen, and a means for detoxifying oxygen, for the anaerobe in reciprocal exchange for a hospitable environment for the aerobe in which to thrive. Since then, mitochondria have become integrated into the crux of cellular function.¹ Critical for the maintenance of homeostasis, the mitochondrion functions as a source of raw materials for amino acid and heme biosynthesis, a buffering system for Ca²⁺, a sensor for O₂, a gatekeeper for apoptotic signaling, a source of reactive oxygen species (ROS), and a heat source for certain vertebrates (brown adipose tissue; BAT).² Their most important role, and the one for which they are best known, is the production of ATP through oxidative phosphorylation. It is through this role that mitochondria have shaped our physiology by facilitating the development of complex cardiovascular, digestive, and hepatic systems to efficiently transport O₂ and nutrients to cells and to remove waste generated through metabolic reactions. This article is an introduction to basic mitochondrial function and will touch on a few of the many important roles mitochondria play in cellular biology.

The mitochondrion is well known for its ability to efficiently convert metabolic byproducts into ATP. This conversion occurs by the electron transport chain (ETC) through the oxidation of reducing equivalents generated during glycolysis, the tricarboxylic acid (TCA) cycle, and β-oxidation. The ETC consists of four primary complexes (I-IV), which, through a series of redox reactions, facilitate the reduction of O₂ and the translocation of protons from the matrix to the intermembrane space. Since the inner mitochondrial membrane is impermeable, these translocated protons establish a gradient, or membrane potential (Δᴪ_M), to be utilized by the ATP synthase. This proton gradient is essential for the synthesis of ATP and correlates directly with the rate of O₂ consumption (OCR) by the ETC. The relationship between the ETC and ATP synthesis is linked by Δψ_M, which is described using the term “coupled.” Compounds that dissipate the Δψ_M, and as a result, increase OCR (e.g., FCCP), are classified as uncouplers, whereas other compounds that dissipate Δψ_M by preventing OCR or the translocation of protons by the ETC, are classified as inhibitors. Both uncouplers and inhibitors can negatively affect the efficiency of the mitochondrion through the dissipation of Δψ_M.

Mitochondrial uncoupling occurs naturally in BAT, which derives its color from the excess of mitochondria. In mammals, BAT is known to induce non-shivering thermogenesis due to the expression of Uncoupling Protein (UCP) 1, which uses Δψ_M to generate heat, resulting in high OCR, with little ATP production. Three types of UCPs, appropriately named 1, 2, and 3, have currently been identified. Whereas UCP1 is expressed only in BAT, the other two are expressed in a variety of tissue types.^3,4 UCPs function not only to generate heat, but also to regulate Δψ_M. Activation of UCPs has been shown to correlate with oxidative stress and ROS. All UCPs are inhibited by guanosine diphosphate (GDP) whereas genipin specifically inhibits UCP2. In addition to UCPs, mitochondria also possess a basal proton leak, which helps to prevent dielectric breakdown due to hyperpolarization. For more information on proton leak see publications from Martin Brand’s group.^5,6

While providing an energy intermediate to drive ATP synthesis, Δψ_M also influences the generation of ROS. To be more precise, higher Δψ_M results in decreased OCR, which in turn, leads to increased levels of ROS generation. The relationship between Δψ_M and ROS generation correlates to the effect of Δψ_M on OCR. Since OCR is proportional to the rate of electron transfer (4e^-/O₂), OCR dictates the redox status of the ETC. Because of this, a slower OCR results in a more reduced ETC, which is more likely to produce ROS at one of the ROS generating sites. These sites of ROS generation include (but are not limited to) complexes I, III, and the electron transport flavoprotein, which is involved in β-oxidation. The production of ROS by the ETC depends on both the concentration of electron donors (R^•) and the concentration of electron acceptors (e.g., O₂).⁷ Under conditions where OCR is high (e.g., actively phosphorylating mitochondria or in uncoupled mitochondria) the ETC is more oxidized, therefore making it thermodynamically less favorable for ROS production to occur.^8-13 However, when OCR is low (e.g., non-phosphorylating or in mitochondria with high Δψ_M) and not limited by O₂, ROS generation is high, due to a more reduced ETC. Under conditions where O₂ is limiting (e.g., hypoxia), the potential to generate ROS is high, yet, in isolated mitochondrial systems, generation of ROS does not increase due to a lack of an electron acceptor.⁹ Paradoxically, a burst of mitochondrial ROS has been shown to occur under hypoxic conditions aiding in the stabilization of the hypoxia inducible factor-1 (HIF-1).¹⁴

The chemiosmotic proton gradient generated by the ETC is the driving force behind virtually all mitochondrial function. This Δψ_M, which provides the driving force for ATP synthesis, heat generation, and ROS production, also allows mitochondria to function as cellular Ca²⁺ buffers. Using specialized Ca²⁺ transporters (Ca²⁺ uniporter [Ca²⁺ uni] and rapid mode of Ca²⁺ uptake [RaM]), Ca²⁺ is transported into the mitochondrial matrix, along with water, resulting in swelling of the inner mitochondrial membrane.¹⁵ The ability of mitochondria to buffer Ca²⁺ is critical for nominally functioning myocytes and neurons. However, a careful balance must be maintained. Should the mitochondria take up excess Ca²⁺ (as occurs during ischemia-reperfusion injury), the inner mitochondrial membrane will become permeable via opening of the mitochondrial permeability transition pore (mPTP). An open mPTP results in instantaneous mitochondrial depolarization, release of cytochrome c, and ultimately cell death. Opening of the mPTP can also be triggered by oxidative stress. In small amounts, ROS generation can regulate Δψ_M by activating UCPs, whereas large amounts can overwhelm antioxidant defenses and result in the opening of mPTP. For a more detailed review on the balance between Ca²⁺ and ROS, see Brookes et al.¹⁶

This dynamic balance between Ca²⁺ and ROS sensitizes mitochondria to diseases affecting oxidant levels, glucose levels, and ion homeostasis. While many of these diseases are the focus of the pharmaceutical industry, some of the recent compounds developed to treat these diseases also have adverse effects on mitochondrial function. One such compound is the diabetes drug metformin, which inhibits complex I. Effects of other drugs range from inhibiting the ETC, inhibiting ATP synthase, or a mild to severe uncoupling, thus making the mitochondrion susceptible to drug induced toxicity.

While mitochondria are critical in powering a number of cellular processes, they are also uniquely adapted to aid the cell in functions that are independent of ATP synthesis. The recent edition of Bioenergetics 4 is a comprehensive resource for describing these detailed and complex mechanisms.¹⁷ With further research, we can establish a better understanding of these unique organelles which are essential for maintaining biological homeostasis.

Illustration of basic mitochondrial functions outlined in the text. The ETC is shown producing ROS and generating a proton gradient through the reduction of O₂. This is then utilized by the F₁F_o ATP synthase (complex V), to generate ATP from ADP and P_i. The mitochondrial membrane potential is indicated by Δψ_M with + or – showing the respective charge. IMM inner mitochondrial membrane • OMM outer mitochondrial membrane • CypD cyclophilin D P i phosphate, and its respective transporter • ANT adenine nucleotide translocase

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