Class 9: Cellular Respiration Rate Experiments & Photosynthesis–Respiration Integration

Goal of the lab session: quantitatively measure the rate of cellular respiration (specifically, O(_2) consumption) in isolated mitochondria.
Source of mitochondria: large volumes (≈1–2 L) of Chlamydomonas (“clammy”) cells are harvested.
- Organelles other than mitochondria (chloroplasts, ER, etc.) are discarded.
- Cells are carefully ground, centrifuged, and washed to remove extraneous solutes.
- Desired final product: “juicy, fresh, intact mitochondria” that neither lyse nor swell.
Mitochondria are suspended in an isosmotic, pH-buffered solution and introduced into a sealed oxygen-electrode cuvette.
- Electrode continuously records dissolved O(2) concentration; slope of O(2) decline = respiration rate.
- A small injection port allows sequential, cumulative additions of test compounds (substrates, inhibitors, uncouplers, etc.).

Each 2-min window (0–2, 2–4, 4–6 min …) is analyzed separately.
Instructor’s scoring rubric (“Twinkies challenge”):
- Straight lines only → report slope (steep = fast, flat = slow).
- Do not draw curved transitions; conceptual simplification.
- All previously added compounds remain present; concentrations are assumed non-limiting.

0 – 2 min: Add washed mitochondria only
- Observation: no significant change in O(_2).
- Rationale: washing removes endogenous NADH/FADH(_2); e⁻ transport chain (ETC) starved of electrons.
2 – 4 min: Inject NADH
- Observation: slope becomes negative (declining O(_2)), but relatively modest.
- Mechanistic note: NADH donates e⁻ to Complex I → proton pumping begins, but the resulting ∆pH/∆Ψ builds quickly because protons cannot return (see point 3).
4 – 6 min: Add ADP + P(_i)
- Observation: slope becomes steeper (faster O(_2) consumption).
- Key concept: Respiratory control
  - ATP synthase (Complex V) is a gated channel; opens only when its substrates (ADP + P(_i)) are available.
  - Opening dissipates the proton gradient ((\Delta p)); ETC no longer works against a large back-pressure → e⁻ flow and O(_2) reduction accelerate.
6 – 8 min: Add an uncoupler (e.g., 2,4-dinitrophenol)
- Observation: slope becomes the steepest of all (maximal respiration rate).
- Uncoupler shuttles protons across the inner mitochondrial membrane independent of ATP synthase → completely abolishes (\Delta p).
- Consequences:
  - ETC runs at its uninhibited chemical limit (rates can be 2- to 5-fold higher).
  - Virtually no ATP produced by oxidative phosphorylation; energy released as heat.
- Clinical/fitness anecdote: overdosing on uncouplers causes hyper-metabolism and death from ATP deficiency.

Oxygen as Terminal Electron Acceptor
- Overall reduction: $O2 + 4\,e^- + 4\,H^+ \rightarrow 2\,H2O$
- Falling O(_2) concentration directly reflects electron-transport activity.
Respiratory Control Logic
- Cellular objective: oxidize NADH/FADH(_2) only when the resulting proton motive force (PMF) can be harnessed for ATP synthesis.
- Prevents futile oxidation when ADP is scarce.
Rate-limiting Factors
- Substrate availability (NADH, FADH(_2)).
- PMF magnitude (back-pressure).
- Permeability changes (e.g., uncouplers).

ATP produced inside the chloroplast never leaves; it cycles only between light reactions and the Calvin cycle.
Exportable product is glyceraldehyde-3-phosphate (G3P):
- One G3P (3-C) generated every 3 Calvin-cycle turns.
- Transported to cytosol via a dedicated G3P exporter.
Cytosolic fates of G3P:
1. Energy: enters glycolysis → pyruvate → mitochondrion → citric acid cycle + OXPHOS → lots of ATP.
2. Biosynthesis: carbon backbone for fatty acids, amino acids, cell wall polysaccharides, etc.
Mass balance insight: if all G3P were fully respired (3 CO(2) out for 3 CO(2) in) no net biomass increase would occur. Growth requires diverting a fraction of G3P into anabolic pathways.

Strict autotrophy (light + CO(_2) only) supports growth but is photon-limited.
Heterotrophy is possible because the alga possesses an acetate transporter (but no glucose transporter).
- Environmental acetate (CH(_3)COO(^-)) enters, becomes acetyl-CoA → TCA cycle → ATP and biosynthetic precursors.
Mixotrophic growth (TAP medium + light) combines photosynthetic G3P production with acetate metabolism → fastest doubling times (~10 h in lab culture).

The “Twinkies challenge” is a motivational classroom game; prizes are low-stakes ($≈3.49 box).
Uncoupler misuse (e.g., in bodybuilding) illustrates ethical concerns: dangerous metabolic manipulation for aesthetic goals.

Proton-to-ATP coupling via ATP synthase ≈ 3 H(^+)/ATP (context-dependent; not tested here).
Typical uncoupling experiment timing: 2-min intervals; interpret slope only within each window.
Overall aerobic glucose oxidation: $C6H{12}O6 + 6\,O2 \rightarrow 6\,CO2 + 6\,H2O + \text{~30 – 32 ATP}$ (mentioned qualitatively).

Be able to predict how respiration rate changes when any one of the following is added to isolated mitochondria: NADH, FADH(2), ADP + P(i), oligomycin (ATP-synthase blocker), rotenone (Complex I inhibitor), an uncoupler.
Always ask: “What limits e⁻ flow right now—substrate supply, PMF, or enzyme inhibition?”
Remember that growth requires both energy and carbon skeletons; tracing carbon flux (G3P vs. CO(_2)) helps explain biomass accumulation.