(a)(i) Write down the reactions that produce NADH during Krebs cycle. (10 marks) (a)(ii) Explain the role of proton gradient in…

(a)

(i) Reactions that produce NADH in the Krebs cycle

In the mitochondrial matrix, each turn of the cycle oxidises one acetyl group and reduces three NAD⁺ to NADH (plus one FAD to FADH₂ and one GTP/ATP by substrate-level phosphorylation). The three NADH-producing steps are:

1. Isocitrate → α-ketoglutarate (oxidative decarboxylation), catalysed by NAD⁺-dependent isocitrate dehydrogenase, the main rate-limiting enzyme of the cycle (activated by ADP and Ca²⁺, inhibited by ATP and NADH). The enzyme-bound intermediate is oxalosuccinate, and the first CO₂ is released: Isocitrate + NAD⁺ → α-ketoglutarate + CO₂ + NADH + H⁺
2. α-Ketoglutarate → succinyl-CoA (oxidative decarboxylation), catalysed by the α-ketoglutarate dehydrogenase complex, which needs thiamine pyrophosphate, lipoic acid, CoA, FAD and NAD⁺ (like pyruvate dehydrogenase); the second CO₂ is released: α-Ketoglutarate + NAD⁺ + CoA-SH → succinyl-CoA + CO₂ + NADH + H⁺
3. L-Malate → oxaloacetate (dehydrogenation), catalysed by malate dehydrogenase; though unfavourable (ΔG°′ ≈ +29.7 kJ/mol), it proceeds because oxaloacetate is removed rapidly by citrate synthase: L-Malate + NAD⁺ ⇌ oxaloacetate + NADH + H⁺

The succinate dehydrogenase step (succinate → fumarate) reduces FAD, not NAD⁺. Counting the pyruvate dehydrogenase reaction that feeds the cycle, one pyruvate yields four NADH, so one glucose gives eight NADH in the mitochondrion (plus two from glycolysis).

(ii) Role of the proton gradient in oxidative ATP synthesis

Peter Mitchell's chemiosmotic hypothesis (1961; Nobel Prize 1978) explains how electron transport is coupled to ATP synthesis through a proton gradient across the inner mitochondrial membrane.

• Generating the gradient: as electrons pass from NADH to O₂, complexes I (NADH dehydrogenase), III (cytochrome bc₁) and IV (cytochrome c oxidase) pump protons from the matrix into the intermembrane space: about 4, 4 and 2 H⁺ respectively, or 10 H⁺ per NADH and 6 per FADH₂ (which enters at complex II, not a pump). The inner membrane is impermeable to H⁺, so the gradient is stored.
• Proton-motive force: the gradient has an electrical component (membrane potential Δψ, about 150–180 mV, matrix negative) and a chemical component (ΔpH, about 0.5–1 unit, matrix alkaline), together about 200 mV.
• ATP synthase: protons return to the matrix only through Fₒ, the membrane part of the F₀F₁-ATP synthase. Their passage turns the ring of c-subunits and the attached central stalk (γ and ε). The rotating γ subunit changes the conformation of the three catalytic β subunits in F₁ in turn through open, loose and tight states (Boyer's binding-change mechanism, confirmed by Walker's structure), so ADP + Pᵢ are bound, ATP is formed and ATP is released; the peripheral stalk holds F₁ stationary.
• Stoichiometry: with 8 c-subunits in mammals, about 8 H⁺ drive three ATP, and one more H⁺ is used to bring in Pᵢ and exchange ATP for ADP, giving P/O ratios of about 2.5 for NADH and 1.5 for FADH₂.
• Evidence: ATP synthesis requires an intact membrane; an artificial pH gradient drives ATP synthesis in the dark (Jagendorf's chloroplast experiment); uncouplers such as 2,4-dinitrophenol and thermogenin (UCP1) in brown fat dissipate the gradient so that oxidation continues as heat without ATP; and oligomycin blocks Fₒ and stops both ATP synthesis and respiration.

(b) Lymphocytes, monocytes and neutrophils

Lymphocytes:

• Features: about 20–40% of leucocytes; small (7–10 µm) or large (up to 15 µm) cells with a large, round, densely stained nucleus and a thin rim of pale basophilic, agranular cytoplasm (large granular lymphocytes are mostly NK cells). They arise from lymphoid progenitors in the bone marrow; T cells mature in the thymus. Lifespan ranges from days to many years for memory cells.
• Functions: they carry out specific (adaptive) immunity with memory. B cells bear surface antibody and differentiate into plasma cells that secrete antibodies (humoral immunity) and into memory B cells. CD4⁺ helper T cells secrete cytokines that direct B cells, macrophages and CD8⁺ cells; CD8⁺ cytotoxic T cells kill virus-infected and tumour cells by perforin–granzyme and Fas–FasL; regulatory T cells maintain tolerance. Natural killer cells kill infected or tumour cells lacking MHC I without prior sensitisation (innate).

Monocytes:

• Features: about 2–8% of leucocytes; the largest leucocytes (12–20 µm), with an eccentric kidney- or horseshoe-shaped nucleus with lacy chromatin and abundant grey-blue cytoplasm with fine azurophilic granules and vacuoles. They circulate for 1–3 days before entering tissues.
• Functions: in tissues they become macrophages (Kupffer cells in the liver, alveolar macrophages, microglia derive largely from embryonic precursors) and some dendritic cells. They phagocytose microbes, dead cells and debris; process antigens and present them on MHC class II to helper T cells; secrete IL-1, TNF-α and IL-6 (fever and inflammation); and help tissue repair. Chronic inflammation and granulomas (as in tuberculosis) are macrophage-dominated.

Neutrophils:

• Features: the most numerous leucocytes (about 40–70%); 10–12 µm, with a nucleus of 3–5 lobes joined by thin strands (hence "polymorphonuclear"; female cells may show a drumstick) and faintly staining cytoplasm with primary (azurophilic) granules containing myeloperoxidase, elastase and defensins and secondary (specific) granules containing lactoferrin and lysozyme. They circulate for less than a day before entering tissues, where they live 1–2 days.
• Functions: the first cells to arrive at sites of acute bacterial infection. They roll along endothelium (selectins), adhere (integrins), migrate out (diapedesis), and move by chemotaxis towards IL-8, C5a and bacterial peptides; they phagocytose opsonised bacteria and kill them by the respiratory burst (NADPH oxidase producing superoxide and, with myeloperoxidase, hypochlorous acid) and by granule enzymes; they can also release neutrophil extracellular traps (NETs) of DNA and proteins. Dead neutrophils form pus; a rise (neutrophilia) indicates acute bacterial infection.

In short, neutrophils are short-lived first responders in innate immunity, monocytes are longer-lived phagocytes and antigen-presenting cells linking innate and adaptive immunity, and lymphocytes carry out specific adaptive immunity and memory.

(c) Development of the heart in mammals

The heart is the first functional organ of the embryo. In humans it begins to beat around day 22, and the main steps (with mouse equivalents) are:

• Cardiogenic field: in the third week (about day 18–19; mouse E7.5), cells of the splanchnic layer of lateral plate mesoderm, induced by the endoderm (BMP, FGF, with Wnt inhibition), form a crescent-shaped cardiogenic area cranial to the neural plate. It contains a first heart field, which forms mainly the left ventricle and parts of the atria, and a second heart field, which later adds the right ventricle, the outflow tract and much of the atria. Transcription factors Nkx2.5, GATA4 and Tbx5 mark these cells.
• Heart tube: with lateral folding of the embryo, the paired endocardial tubes are brought to the midline and fuse (about day 21–22; mouse E8.0) into a single tube. It has an inner endocardium, a middle layer of cardiac jelly and an outer myocardium, and it lies in the pericardial cavity, suspended by the dorsal mesocardium.
• Primitive chambers: from cranial (arterial) to caudal (venous) end, the tube shows the truncus arteriosus, bulbus cordis, primitive ventricle, primitive atrium and sinus venosus, which receives the vitelline, umbilical and common cardinal veins.
• Looping: from about day 23 to 28, the tube elongates by addition of second-heart-field cells and bends to the right (dextral or D-looping) into an S-shape. The bulbus cordis and ventricle come to lie ventral and caudal, while the atrium and sinus venosus move dorsal and cranial; this brings the chambers into their final relative positions. Left–right signals (Nodal, Pitx2) control the direction of looping.
• Atrial septation (weeks 4–6): the septum primum grows down from the roof of the atrium towards the endocardial cushions, leaving the foramen primum; before this closes, perforations in its upper part form the foramen secundum. A thicker muscular septum secundum then grows to the right of the septum primum, leaving an opening, the foramen ovale. The septum primum acts as a flap valve that lets blood pass from right to left atrium before birth and closes against the septum secundum after birth, when left atrial pressure rises.
• Atrioventricular canal: dorsal and ventral endocardial cushions fuse to divide the canal into right and left openings, where the tricuspid and mitral valves form.
• Ventricular septation: a muscular interventricular septum grows up from the floor of the primitive ventricle, leaving an interventricular foramen that is closed by the membranous septum, formed from the endocardial cushions and the conotruncal ridges, by about week 7.
• Outflow tract: neural-crest-derived truncal and bulbar ridges fuse in a spiral to form the aorticopulmonary septum, dividing the truncus into the ascending aorta and pulmonary trunk; the semilunar valves form from the ridges.
• Sinus venosus: the right horn is absorbed into the right atrium (the smooth sinus venarum), the left horn becomes the coronary sinus, and the pulmonary veins are incorporated into the left atrium.

By about week 8 (mouse E14.5) the four-chambered heart is formed. Before birth the foramen ovale and ductus arteriosus bypass the lungs; they close after birth to separate the pulmonary and systemic circulations. Defects in these steps cause atrial and ventricular septal defects, transposition of the great arteries and tetralogy of Fallot.