Evolutionary Pressure on Mitochondrial Cytochrome b Is Consistent with a Role of CytbI7T Affecting Longevity during Caloric Restriction
McClellan DA (2009) Evolutionary Pressure on Mitochondrial Cytochrome b Is Consistent with a Role of CytbI7T
Affecting Longevity during Caloric Restriction. PLoS ONE 4(6): e5836. doi:10.1371/journal.pone.0005836
Evolutionary Pressure on Mitochondrial Cytochrome b Is Consistent with a Role of CytbI7T Affecting Longevity during Caloric Restriction
Wesley A. Beckstead 0 1
Mark T. W. Ebbert 0 1
Mark J. Rowe 0 1
David A. McClellan 0 1
Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America
0 a Current address: Oncogenomics Section, Pediatric Oncology Branch, Advanced Technology Center, National Cancer Institute, Gaithersburg, Maryland, United States of America b Current address: Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, United States of America c Current address: Bigelow Laboratory for Ocean Sciences , West Boothbay Harbor, Maine , United States of America
1 1 Department of Biology, Brigham Young University , Provo , Utah, United States of America, 2 Department of Nutrition , Dietetics, and Food Science , Brigham Young University , Provo, Utah , United States of America
Background: Metabolism of energy nutrients by the mitochondrial electron transport chain (ETC) is implicated in the aging process. Polymorphisms in core ETC proteins may have an effect on longevity. Here we investigate the cytochrome b (cytb) polymorphism at amino acid 7 (cytbI7T) that distinguishes human mitochondrial haplogroup H from haplogroup U. Principal Findings: We compared longevity of individuals in these two haplogroups during historical extremes of caloric intake. Haplogroup H exhibits significantly increased longevity during historical caloric restriction compared to haplogroup U (p = 0.02) while during caloric abundance they are not different. The historical effects of natural selection on the cytb protein were estimated with the software TreeSAAP using a phylogenetic reconstruction for 107 mammal taxa from all major mammalian lineages using 13 complete protein-coding mitochondrial gene sequences. With this framework, we compared the biochemical shifts produced by cytbI7T with historical evolutionary pressure on and near this polymorphic site throughout mammalian evolution to characterize the role cytbI7T had on the ETC during times of restricted caloric intake. Significance: Our results suggest the relationship between caloric restriction and increased longevity in human mitochondrial haplogroup H is determined by cytbI7T which likely enhances the ability of water to replenish the Qi binding site and decreases the time ubisemiquinone is at the Qo site, resulting in a decrease in the average production rate of radical oxygen species (ROS).
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 [10,11].
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 [12,13].
Further, antioxidants have been shown not to prolong lifespan (e.g.
[14,15]). 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
ROSsignal 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 [8,10,18]. 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
(19201940) 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.
Figure 1 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.
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 192040 longevity representing
historical calorie restriction, and 196080 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.
Materials and Methods
Haplotype and Longevity Data Collection
The haplotypes and life spans of individuals who lived between
1870 and the present were collected from Family Tree DNA
(http://www.familytreeDNA.com) and the Sorenson Molecular
Genealogy Foundation (http://www.smgf.org). The female
individuals whose haplogroup was identified from these databases
were used to identify maternally related pedigrees from Family
Search (http://www.familysearch.org). Data for 737 individuals
from Haplogroup H and 890 individuals from Haplogroup U were
collected and evaluated.
Individuals were included if they met the following criteria.
First, only females were included to avoid the differences between
female and male longevity. Second, individuals were included if
they died in North America. This criterion was chosen in order to
increase the probability that individuals included in this study lived
under somewhat similar caloric environments. Finally, the
individuals were included only if they died at or after the age of
60. This was done to increase the probability that included
Figure 1. Haplogroup relationship for H and U. The mitochondrial SNPs that separate them are shown. Non-synonymous SNPs are shown in
red. RNA gene mutations are shown in black. The data was obtained from MITOMAP (http://www.mitomap.org) [41,42]. The only protein-coding SNP
that separates the two haplogroups and that is therefore analyzed in this study is T14766C, which results in an amino acid substitution of a threonine
for an isoleucine at site 7 in cytochrome b.
individuals died from age related factors including degenerative
disorders rather than by accidents or other unrelated causes.
The human population has undergone dramatic shifts in caloric
intake during different time periods throughout the last 200 years.
In order to elucidate the impact of caloric intake on longevity,
individuals were grouped together by the time period in which
they died. Table 1 shows the number of individuals from each of
the two haplogroups H and U in each time period.
Biochemical and Evolutionary Correlation
As shown in Figure 1, haplogroup H is separated from
haplogroup U and other haplogroups by mitochondrial SNP
T14766C, which results in an amino acid substitution of a
threonine for an isoleucine at site 7 in cytb, thus possibly affecting
protein complex III and the ETC. In order to computationally
generate information about the possible physicochemical effects of
this SNP on cytb, respiratory chain efficiency, and longevity in
Haplogroup H individuals, the analytical program TreeSAAP
 was used to examine the following:
N The effect(s) cytbI7T may have on the physicochemical
properties of the N-terminal region of the cytb protein.
N The naturally occurring effects of selection on the
physicochemical properties of the N-terminal region of
the cytb protein over the phylogenetic history of mammals
to establish a context within which to interpret the
broader scope of the human SNP cytbI7T.
The details of these two steps will be discussed in the remainder
of this section.
TreeSAAP was originally developed to detect molecular
adaptation due to natural selection across a protein sequence by
statistical analysis of the amino acid substitutions across a
phylogenetic tree. This adaptation is expressed in terms of amino
acid property changes. TreeSAAP measures the physicochemical
magnitude of amino acid substitutions and indicates which amino
acid properties have likely been affected by natural selection
during the evolutionary process.
As the first step, we modified the use of TreeSAAP, allowing the
program to analyze the physiochemical changes produced by a
single amino acid substitution . TreeSAAP was thus used to
detect the radical physicochemical shifts that are produced by a
mutation substituting a threonine for an isoleucine at site 7 in cytb
of mitochondrial complex III. TreeSAAP was implemented by
grouping changes into one of eight magnitude categories, 1 being
the most conservative and 8 being the most radical. In this study
we chose to focus on amino acid property changes of category 6, 7,
and 8 because they unambiguously indicate a significant change in
the protein .
In the second step, the entire protein-coding portion of the
mitochondrial genome (13 complete coding sequences) was
collected for 107 mammalian species, including sequences from
all major mammalian lineages, from GenBank (Supplementary
Table S1). These sequences were aligned and used for
phylogenetic analysis (Supplementary Figure S1). The cytb gene sequences
for the 107 mammalian species and the phylogenetic tree created
from all 13 mtDNA genes were used as input for TreeSAAP as in
Chamala et al. .
We used TreeSAAP to gain a historical context of the amino
acid property changes at site 7 in cytb. Specifically, the property
changes that TreeSAAP detected in step 1 of the analysis were
correlated with the same properties TreeSAAP detected to be
under natural selection throughout mammalian evolution. The
overall purpose of this second step is to establish context for
properly interpreting the results of step one. By estimating the
naturally occurring pattern of adaptation, the effects of SNPs may
be compared to the location and effects of extant genetic variation
and historical adaptations within the broader taxonomic group.
When a SNP fails to share characteristics with the historical
adaptations of the group, that SNP is more likely have a
detrimental effect. Conversely, if a SNP has much in common
with the adaptations of the group, that SNP may have a similar
adaptive effect .
Difference in Longevity between H and U
The mean age at death was calculated for each time period
cohort of individuals. To test for statistical difference between
Haplogroup H and U, a two-tailed t-test assuming equal variances
was performed on the means. Figure 2 shows the mean age at
death for each time period cohort. We see an expected general
increase in longevity during the 20th century in both haplogroups.
Before 1920 there is no significant difference between the longevity
of individuals in haplogroup H and U. During the caloric
restriction of the Great Depression, 19201940, haplogroup H
shows significant increase in longevity compared to haplogroup U
(mean difference = 2.6 years, p = 0.02).
This significant difference during caloric restriction is further
illustrated by a survival curve of individuals in the two haplogroups
during this two decade time period (Figure 3). Following 1940,
there is little difference in longevity between Haplogroup H and U
individuals. This lack of difference continues to include recent
years of caloric over-consumption.
CytbI7T TreeSAAP Results
Table 2 shows the TreeSAAP results for the single substitution
cytbI7T. TreeSAAP indicated that substituting a threonine for an
isoleucine at site 7 in cytb results in radical shifts in seven amino
acid properties. The large number of properties of magnitude 68
(radical to extremely radical changes) that are associated with this
polymorphism suggests a radical change in the resulting protein.
These properties will be described in detail.
Three properties relate to the level of hydrophilicity in the region.
In general, the amino acid property Surrounding Hydrophobicity
refers to the tendency for the region around the amino acid site in
question to interact with water. In the case of cytbI7T, there is a
radical decrease of magnitude 8 in this property, indicating that the
region surrounding site 7 becomes less hydrophobic and more
hydrophilic by introducing a threonine instead of an isoleucine.
This biochemical shift is likely the result of the hydroxyl group on
the R-group of threonine that may easily interact with water
molecules. Isoleucine is hydrophobic and thus lacks this ability.
Hydrophobicity is similar to the property Hydropathy, which also
decreases due to the polymorphism I7T.
Equilibrium Constant deals with the ability of any ionizable
functional group of the residue to dissociate and make an ion [27
29]. TreeSAAP indicates that cytbI7T increases this property by a
moderately radical (magnitude 6) change. An increased
equilibrium constant for the ionization of COOH would indicate a more
product-driven reaction. This would, as in the other properties,
make the region more water-soluble and hydrophilic. As will be
noted, this may be of greatest importance regarding reduced ROS
production and increased longevity during calorie restriction.
Figure 2. The mean age at death of individuals grouped by time period. Tests for statistical difference were performed using a two-tailed
ttest. Haplogroup U is labeled in red and Haplogroup H is labeled in yellow. Haplogroup H shows significantly increased longevity during 19201940.
The other four properties relate to the level of compactness in
the region. The amino acid properties Average Number of
Surrounding Residues and Buriedness measure how compact
and buried the amino acid site is. TreeSAAP indicated that
cytbI7T decreases both of these properties with a magnitude 6
change. With a decrease in these properties, we can imagine a
region around site 7 that is more open and free to interact (as
opposed to compact).
This conclusion is further supported by the decrease in the
amino acid property Long-range non-bonded energy resulting
from cytbI7T. This property describes the interactions between
molecules that are not directly in contact with one another (such as
Van der Waals interactions), affecting the stability of molecules
involved. It has been shown that the structures in globular proteins
are influenced not only by local, bonding interactions, but also by
long-range interactions [29,30]. TreeSAAP indicated a radical
decrease in this property of magnitude 6. A decrease in
Longrange non-bonded energy indicates a decrease in stability in this
residue and surrounding residues, further suggesting that the
region around site 7 is more open, less globular, and less compact
due to this polymorphism.
The solvent accessible surface of a protein is the region where
solvent and solutes interact with the protein . The amino acid
property Solvent Accessibility Reduction Ratio is defined as the
ratio of the solvent accessible surface area of a residue in the native
state to that of the residue in an extended tri-peptide (Ala-X-Ala)
All of these property changes involve either an increase in
hydrophilicity or a decrease in compactness in the region
surrounding site 7 in cytb among haplogroup H individuals.
Mammalian Evolution TreeSAAP Results
The second part of our analysis involved the use of TreeSAAP
across the cytb sequences and phylogenetic tree of 107
Equilibrium Constant (Ionization of COOH)
Average Number of Surrounding Residues
Long-Range Non-Bonded Energy
Solvent Accessible Reduction Ratio
The amino acid property changes, the radicality of the change, and the direction of the change are shown.
mammalian species from all major mammalian lineages in order
to estimate a historical evolutionary context of the amino acid
properties that have been under selection for radical change at site
7 in cytb. Particularly, the amino acid properties that are changed
by the cytbI7T polymorphism itself (Table 2) were inspected in the
TreeSAAP results of 107 mammalian cytb sequences.
Figure 4 shows the TreeSAAP results across cytb that are
consistent with the amino acid properties found by running the
individual cytbI7T polymorphism through TreeSAAP. Though
TreeSAAP found radical shifts in 7 amino acid properties while
analyzing the individual cytbI7T polymorphism, only six of these
were significantly affected in the mammalian data set TreeSAAP
did not detect evidence of adaptation for the property Average
Number of Surrounding Residues.
The peaks in the graphs of Figure 4 represent radical
changes in those properties during phylogenesis at particular
amino acid sites. Site 7 in cytb, where the polymorphism that
distinguishes haplogroup H occurs, are marked in each graph.
There are no peaks in any of these graphs at site 7, though
there have been naturally occurring radical changes in these
properties elsewhere along the sequence. This indicates that the
7 properties affected by cytbI7T have been highly conserved
and stable at this site throughout the evolution of these 107
TreeSAAP results suggest that the region around site 7 likely
was not a site of adaptation in hydrophilicity and compactness
throughout mammalian evolution. This result, however, does not
preclude positive selection at this site in the human lineage.
Threonine was either fixed or maintained at cytb site 7 until a T7I
mutation occurred that led to an isoleucine polymorphism in
primates. Subsequent primates, including most human
mitochondrial haplogroups, still exhibit isoleucine at site 7. Relatively
recently, the human substitution cytbI7T occurred, forming
haplogroup H. Radical physicochemical shifts in haplogroup H
restore the historical pre-primate character state, suggesting that
cytbI7T may have a positive effect. Given its location within
protein complex III and the radical nature of the physicochemical
effect, we are led to conclude that cytbI7T was likely advantageous
relative to the precise efficiency of complex III and the respiratory
chain that is linked to increased longevity during calorie
The I7T polymorphism analyzed in this study is located in the
cytb protein subunit. Cytb is encoded by mitochondrial DNA and
is located centrally in complex III (cytochrome bc1 complex) of the
ETC. Complex III is a dimer enzyme embedded in the inner
membrane of the mitochondria. The complex couples electron
transfer from ubiquinol to cytochrome c (cytc) with proton
translocation across the membrane to contribute to an increased
proton concentration in the intermembrane space of the
mitochondria . The resulting proton gradient drives ATP
synthesis via oxidative phosphorylation by ATPase (protein
The coupling of ubiquinol and cytc is accomplished by the Q
cycle, which involves oxidation of ubiquinol at the Qo binding site
to create ubiquinone, which is then reduced at the Qi binding site
. Both of these binding sites are located in the protein
subunit cytb situated within the hydrophobic center of the inner
Figure 4. Mammalian evolution TreeSAAP results for the cytochrome b protein subunit. Each graph shows evolutionary selection for a
given amino acid property across the amino acid sequence of cytb. A z-score above 3.09 indicates radical changes occurring for the given property
throughout the evolution of 107 mammalian species (see Materials and Methods). The amino acid site number 7 where the cytb polymorphism
occurs is annotated. (A) Shows the TreeSAAP results for the property Buriedness. (B) Shows the results for property Equilibrium Constant. (C) and
(D) Show results for Hydropathy and long-Range Non-Bonded Energy respectively. (E) and (F) Depict the graphs for the TreeSAAP results of the
properties Solvent Accessible Reduction Ratio and Surrounding Hydrophobicity.
Figure 5. Cytochrome b within the cytochrome bc1 complex. Only one monomer is shown. The cytochrome b subunit is shown in grey with
surrounding subunits shown in green. The CoQ coenzymes are shown bound in the outside (Qo) and inside (Qi) binding sites. The heme groups that
are involved in the transfer of the electrons between binding sites are also shown. The cytbI7T polymorphism (yellow) is located on the N-terminus
tail of cytb near the Qi site. The three-dimensional coordinates of the complex were obtained from the protein data bank (http://www.pdb.org) 
under the entry 1ntz . The structure was visually rendered with PyMOL .
membrane (Fig. 5). The cytbI7T polymorphism is located on the
N-terminus tail of cytb, near the Qi binding site.
Ubiquinone is reduced to ubiquinol at the Qi binding site
[36,38] (Fig. 6). Two important aspects of this reaction are: 1) the
presence of water and ubiquinone within the binding site, and 2)
water molecules filling the binding site after ubiquinol vacates.
High-resolution structures of protein complex III show that water
is involved in hydrogen bonds between the quinone and
surrounding cytb residues [39,40]. Water is also replenished in
the vacant binding site to replace the H+ used in the reduction
reaction . Therefore, water molecules are an essential
ingredient for the ubiquinone to ubiquinol cycle at the Qi site,
and an initial step in proton translocation across the inner
As mentioned, the CoQ binding sites are embedded within the
globular complex and the hydrophobic inner membrane. Given
this information, we suggest it is not trivial to replenish the
essential H20 molecules in the Qi binding site. A careful look at the
region surrounding the binding site sheds light on this problem.
The three-dimensional structure reveals a hydrophilic region that
could potentially be a channel for water to be shuttled into the Qi
binding site that is embedded within the globular complex in the
membrane (Fig. 7).
The cytbI7T polymorphism lies at the heart of this water
channel and likely has an impact on the ability of water to
replenish the Qi site because threonine may bind a water molecule
with its hydroxyl group, whereas isoleucine lacks this ability.
TreeSAAP indicates that the cytbI7T mutation radically changes
seven amino acid properties that have to do with either increasing
the hydrophilicity or reducing the compactness in the area.
Increasing hydrophilicity and making the area more open and less
globular in the cytb N-terminal region, water will likely be more
attracted to the shuttle area and reach the Qi binding site more
easily. This would increase the efficiency of the binding site and
the Q-cycle overall, thereby decreasing the time that
ubisemiquinone exists at the Qo site, on average decreasing ROS production.
To corroborate this idea, TreeSAAP showed that these seven
amino acid properties have been highly stable and conserved
throughout mammalian evolution in the region surrounding the
cytb N-terminal region prior to the T7I SNP in the basal primate
branch. With the reintroduction of cytbI7T that created the
mitochondrial haplogroup H branch of humans, which is most
prevalent in European populations, these properties are radically
altered and the immediate impact presents itself as an increase in
the potential efficiency of the Q-cycle and the ETC overall,
resulting in increased longevity.
The cytbI7T polymorphism may be viewed in the context of
caloric intake in order to better understand how a more efficient
ETC could affect longevity in haplogroup H individuals. Caloric
intake is essentially the ultimate input to the ETC, and the cytbI7T
polymorphism may react differently with different levels of input.
Haplogroup H individuals have significantly increased longevity
during caloric restriction, but are not significantly different from
haplogroup U individuals during other time periods, even during
recent years of caloric over-consumption. The increased ETC
efficiency of the haplogroup H individuals, which cytbI7T seems
to be responsible for, is most advantageous during caloric
This could be for a variety of reasons. ROS leakage from the
mitochondrial ETC is responsible for signaling apoptosis to the cell
when the cell meets a certain threshold. An increase in Q-cycle
and ETC efficiency, particularly under caloric restriction (sparse
electron input), likely lowers the ROS leakage from the
mitochondria and prevent more cells in certain tissues from
undergoing apoptosis, thus increasing longevity in haplogroup H
individuals. As we see no difference in longevity among haplotype
H and U individuals during caloric over-consumption, this
increased efficiency may not be of any advantage during these
time periods because other factors may override the advantage.
For example, a potential factor could be that of excessive electron
input to the ETC as a result of hyper-calorie intake. Having a
highly reduced ETC due to excessive electrons in the system may
drastically increase the rate of ROS production, thus swamping
the benefit haplogroup H individuals receive from cytbI7T. On
Figure 6. The reduction of ubiquinone to ubiquinol in the Qi binding site in cytochrome b. (A) Shows ubiquinone bound in the Qi pocket
to surrounding cytb residues. A water molecule involved in H-bonding is highlighted in red. The reduction of ubiquinone proceeds from (B) to (D)
with the vacation of ubiquinol shown in (E). (F) Shows the vacant Qi site with replenished water molecules (red) that replace the H+ used in the
reaction. The cycle continues in (G) with another ubiquinone entering the binding site and the process starts again. The figure was adapted from
Kolling et al.  and Crofts .
the other hand, we suggest that during caloric restriction the
benefit from cytbI7T plays a critical role on the precise efficiency
of the ETC and increases longevity in haplogroup H as a result.
Figure S1 The phylogenetic tree of the 107 mammalian
mitochondrial genome sequences (protein-coding portions) used
as input for TreeSAAP.
Found at: doi:10.1371/journal.pone.0005836.s001 (4.45 MB EPS)
Conceived and designed the experiments: WAB MTWE MJR DAM.
Performed the experiments: WAB MTWE MJR DAM. Analyzed the data:
WAB MTWE MJR DAM. Contributed reagents/materials/analysis tools:
MJR DAM. Wrote the paper: WAB MTWE MJR DAM.
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