Mitochondria have long been implicated in the aging process 
. The electron transport chain (ETC), embedded within the inner membrane of the mitochondria, is the major producer of reactive oxygen species (ROS), which are presumed to be the primary agent for cell damage and premature apoptosis, affecting aging and longevity 
. The primary intermediate responsible for producing the ROS superoxide is ubisemiquinone, the coenzyme Q (CoQ) radical produced in complexes I, II and III of the ETC 
. Reducing the production of ubisemiquinone in the ETC has been shown to reduce free radical levels and prolong life span in animals 
The mechanism by which ROS affect aging and longevity has recently come under scrutiny (e.g. 
). For years the paradigm of aging (e.g. 
) has predicted that over time ROS leakage leads to accumulation of mitochondrial DNA (mtDNA) mutations and oxidative damage to the cell. Over the lifespan of an individual, the damage may then lead to premature cell death, followed by organ and tissue failure, which are characteristics of aging and associated degenerative disorders. The paradigm concludes that to avoid cumulative damage over time and increase longevity, antioxidants must be taken to combat oxidative stress on cell components.
Recent studies indicate that these basic assumptions should be revisited. It has been shown that free radical leakage fluctuates according to the signals that ROS themselves produce 
. Further, antioxidants have been shown not to prolong lifespan (e.g. 
). Together, the ROS fluctuations and the lack of an antioxidant effect on longevity indicate that the role of free radicals in aging and longevity is more complex than previously thought.
A theory of aging that accounts for a signaling role for endogenous free radicals in maintaining the metabolic status of the cell has been proposed 
, balancing their role in cellular damage. The underlying principles of this theory of aging include: 1) ROS leakage produces mtDNA mutations; 2) ROS produced by ailing mitochondria also signals cellular apoptosis activities; 3) when a threshold of both ailing mitochondria and ROS signals is reached, the cell prematurely commits to apoptosis followed by organ and tissue failure.
Studies have shown that when a given mutation is found in different species it has varying effects based on the comparative rate of ROS leakage in the species 
. The threshold of ROS signal needed for the cell to commit apoptosis depends on the rate at which ROS are produced from the mitochondria. Intuition might suggest that the threshold of ROS signal needed for the cell to commit to apoptosis is static. However, it appears to be dependent upon the rate of ROS production from the mitochondria, exhibiting a tight correlation between mutations and a ROS-signal apoptotic threshold 
Whether the role of free radicals in aging and longevity involves the toxicity of ROS over time or the important signaling role of ROS in programmed cell death, it is important that studies of longevity turn attention toward mechanisms by which ROS is produced in the respiratory chain of the mitochondria, and how leakage affects the cell and might be reduced.
In this regard, it is also essential to come to an understanding of these mechanisms in the context of caloric intake, since electron input to the ETC may alter ROS production 
. It has been proposed that as mitochondria function in decreased phosphorylating modes, the ETC remains in a more reduced state (maximally occupied with electrons) for longer time periods, increasing the production of ubisemiquinone and ROS, thereby decreasing longevity 
. The major contributor toward a more reduced state is excessive calorie consumption (increased electron input), but other factors can exacerbate the problem as well. For example, the ETC may remain more reduced because of inhibition or dysfunction of ATP production via oxidative phosphorylation, blocking the electron flow of the respiratory chain. Reduced ADP, caused by a lack of physical exercise (during which ADP is not present because ATP is not being used), may also inhibit the turning over of electrons and keep the ETC more reduced. As the electron flow is inhibited, not only do more reactive electrons accumulate, but oxygen levels increase as well. This may increase the probability that backed up electrons and oxygen will react and produce free radicals.
The electron transport chain is composed of protein complexes whose individual protein subunits are encoded in either nuclear or mitochondrial DNA. Nonsynonymous single nucleotide polymorphisms (SNPs) in any of the genes encoding ETC subunits could alter the quality of electron flow or affect CoQ binding sites, and subsequently affect ROS production, aging and longevity.
Mitochondria have maintained a core set of genes that encode essential proteins in the ETC. Nonsynonymous mutations in these genes have the potential to affect the ETC, ROS production and longevity in a way that is dependent upon calorie restriction and/or calorie over-consumption. Throughout the last 150 years there have been dramatic extremes in per capita caloric intake. For example, during the Great Depression (1920–1940) many individuals throughout North America were under extremely restricted caloric intake. In more recent decades there has been an increase in caloric intake toward the other extreme, particularly in North America. If there is a relationship between the redox state of the ETC, longevity, mtDNA mutations and extremes of caloric intake, it could be demonstrated by an analysis of historical longevity within mtDNA haplogroups during extended and continental periods of calorie reduction and over-consumption.
The human population is subdivided into mitochondrial haplogroups. Haplogroups are distinguished by a unique set of mitochondrial SNPs, the nonsynonymous of which are of interest in relationship to their potential effect on the mitochondrial respiratory chain and longevity. Many studies have demonstrated the association of certain mtDNA haplogroups with increased longevity (e.g. 
). We chose to focus on haplogroup H, which is one of the more recent haplogroups, but also now the most prevalent European mtDNA haplogroup, and compare historical longevity in closely related haplogroup U individuals under extremes of caloric intake.
shows the haplogroup relationship with regard to mtDNA mutations between H and U. Haplogroup H is separated from haplogroup U by mitochondrial SNP T14766C, which results in an amino acid substitution of a threonine for an isoleucine at amino acid site 7 in cytochrome b (cytb), which encodes the central catalytic enzyme of the mitochondrial protein complex III (cytochrome bc1 complex) of the ETC.
Haplogroup relationship for H and U.
In this study we first identify the difference in longevity in haplogroups H and U specifically during times of caloric restriction as well as times of calorie over-consumption. This is accomplished by identifying individuals with haplotype H and U from two genetic genealogy databases and collecting longevity information about their maternally related ancestors from the pedigrees in those databases. In addition, many of the ancestors were then found in a family history database, and longevity information was gathered about their extended maternal relatives who share their maternally inherited haplogroups H and U. The longevity data for haplogroups H and U were compared in cohorts of 20 year increments, with 1920–40 longevity representing historical calorie restriction, and 1960–80 and 1980-present representing caloric over-consumption.
Next, we examined the biochemical shift that the polymorphism cytbI7T produces in cytb, protein complex III, and the ETC overall. We then estimate selective pressure on amino acid properties throughout mammalian evolution, particularly at site 7 in cytb, to gain a historical evolutionarily context of this region and a better perspective on how this recent human polymorphism may change the mitochondrial system. Finally, we correlate these data and present a mechanism by which cytbI7T may affect ETC efficiency and ROS production by complex III, and consequently longevity in haplogroup H during a restricted dietary environment compared to an environment of excessive caloric intake.