Passage 3: ATP
Regulation of cellular ATP level is critical for diverse biological processes and may be
defective in diseases such as cancer and mitochondrial disorders. While mitochondria
play critical roles in ATP level regulation, we still lack a systematic and quantitative
picture of how individual mitochondrial-related genes contribute to cellular ATP levels
and how dysregulated ATP levels may affect downstream cellular processes. Advances
in genetically encoded ATP biosensors have provided new opportunities for addressing
these issues. ATP biosensors allow researchers to quantify the changes of ATP levels in
real-time at the single-cell level and characterize corresponding effects at the cellular,
tissue, and organismal level.
Mitochondria are best known as the powerhouses of the cell that produce ATP via
oxidative phosphorylation (OXPHOS). Mitochondrial ATP synthesis involves tricarboxylic
acid (TCA) cycle enzymes, electron transport chain complexes, and ATP synthase, in
which acetyl-coenzyme A (CoA) derived from food molecules is oxidized to produce
ATP. During bioenergetic reactions, mitochondria also produce other physiologically
important molecules such as reactive oxygen species.
Two studies presented a new generation of ATP biosensors. In the first, a circularly
permuted green fluorescent protein (cpGFP) was combined with a bacterial ATPbinding protein to generate a reporter that quantitatively reports cellular ATP to
adenosine diphosphate (ADP) ratio. Because of differential binding affinities to ATP
and ADP, the reporter exhibits different degrees of conformational change in cpGFP
and, thus, different fluorescent signal changes in response to ATP or ADP binding.
In the second study, Imamura and colleagues developed a series of highly sensitive
and selective Förster resonance energy transfer (FRET)-based reporters for cellular
ATP concentrations. FRET-based reporters contain a subunit of a bacterial ATP
synthase fused between cyan fluorescent protein (CFP) and yellow fluorescent
protein (YFP). The reversible binding of ATP leads to conformational changes of
the synthase subunit, resulting in altered spatial proximities between fluorescent
proteins and thus changes in the FRET signal.
Dissecting the regulation and function of ATP at the single-cell level. Adapted from
Zhang et al. (2018).
Correct answer: A. In order to turn FAD into FADH2, there needs
to be an addition of two hydrogen atoms. Therefore, answer choices B and D
are eliminated. Even though this is a half-reaction, there need to be two atoms
of hydrogen in order to add the two hydrogens to FAD. No, we only have answer
choices A and C. In order to determine how many electrons are necessary, we
need to first examine the oxidation states of each species in the reactants and
products. The FAD in the reactants has a neutral charge, indicating that it has an
oxidation number of 0. In FADH2, each of the H+ has a charge of +1. Since there are
two hydrogens, this means that both of them have a combined charge of +2. Since
FADH2 has a net neutral charge, in order to balance this +2 charge, the FAD would
have to have a charge of -2. This indicates an oxidation number of -2 for the FAD.
Since the FAD is going from an oxidation number of 0 to -2, it gains two electrons.
Thus, answer choice A is the correct answer.
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