(a) How can the reaction equilibria and reaction rates be explained by using free energy diagram in a simple enzymatic reaction ?…

(a) Reaction equilibrium and rate from a free-energy diagram

A free-energy diagram plots free energy (G) against the reaction coordinate from substrate (S) to product (P).

Equilibrium: The difference in free energy between S and P, ΔG°′, fixes the equilibrium constant: ΔG°′ = −RT ln K_eq. An enzyme does not change the free energy of S or P, so ΔG°′ and K_eq are the same with or without enzyme. A reaction with negative ΔG°′ is favoured in the forward direction, and the enzyme only helps equilibrium to be reached faster.

Rate: The rate depends on the activation free energy ΔG‡, the height of the barrier between the substrate and the transition state (S‡). The rate constant is k = (k_BT/h)e^(−ΔG‡/RT), so even a small fall in ΔG‡ raises the rate greatly (about 5.7 kJ/mol gives a tenfold rise at 25 °C).

Enzyme pathway: The enzyme binds the substrate to form the enzyme–substrate complex (ES), passes through the enzyme-bound transition state (ES‡) and gives the enzyme–product complex (EP) and then P. Binding the transition state more tightly than the substrate lowers ΔG‡ by means of proximity and orientation, strain, acid–base and covalent catalysis, and the environment of the active site. The diagram shows a lower peak with several smaller barriers and dips for ES and EP. The rate-limiting step is the highest barrier.

The same lowering applies to the reverse reaction, so the forward and reverse rates rise by equal factors and equilibrium is unchanged.

(b)(i) Nitrate and nitrite reduction in leaves

Nitrate is reduced in two steps.

1. Nitrate reductase (cytosol): NO₃⁻ + NADH + H⁺ → NO₂⁻ + NAD⁺ + H₂O. It is a molybdenum-containing flavoprotein (with FAD and heme).
2. Nitrite reductase (chloroplast): NO₂⁻ + 6 reduced ferredoxin + 8 H⁺ → NH₄⁺ + 6 oxidised ferredoxin + 2 H₂O. It contains siroheme and an Fe–S cluster; in roots the reductant comes from NADPH.

Nitrite is toxic, so it is quickly moved to the chloroplast; the ammonium is then incorporated into amino acids by glutamine synthetase and GOGAT.

(b)(ii) Protection of nitrogenase from oxygen and hydrogen

Nitrogenase (Fe protein and MoFe protein) is irreversibly inactivated by O₂ and inhibited by its by-product H₂.

Against oxygen:

• Leghemoglobin in legume nodules binds O₂ with high affinity, keeping free O₂ low but supplying bacteroids for respiration.
• Respiratory protection: Azotobacter has very high respiration, which consumes the oxygen.
• Conformational protection: in Azotobacter, the Shethna (FeSII) protein reversibly binds nitrogenase.
• Heterocysts in cyanobacteria have thick walls and lack PS II, so they do not produce O₂.
• Nodule cortex acts as a diffusion barrier to O₂.

Against hydrogen: an uptake hydrogenase oxidises H₂ back to protons and electrons, which also gives back energy and consumes oxygen, protecting the enzyme.

(c)(i) Biosphere reserves

Biosphere reserves are areas of terrestrial and coastal ecosystems recognised under UNESCO's Man and the Biosphere (MAB) programme, designated by the government and then nominated internationally. They conserve biodiversity and genetic resources, promote sustainable development and support research, monitoring and education.

Zonation: a core zone, strictly protected; a buffer zone around it, where research, education and compatible uses are allowed; and an outer transition zone, with human settlements and sustainable agriculture and resource use. Nilgiri (1986) was the first in India, followed by Nanda Devi, Sundarbans, Gulf of Mannar and others; India has 18 designated biosphere reserves, many in UNESCO's world network. Unlike national parks, they include people, but they depend on community participation and effective zone management.

(c)(ii) Red Data Book

The Red Data Book is a record of threatened species, started by the IUCN (1966). It is based on the IUCN Red List categories: Extinct, Extinct in the Wild, Critically Endangered, Endangered, Vulnerable, Near Threatened, Least Concern, Data Deficient and Not Evaluated. They are assigned using criteria of population size, rate of decline, range and extinction probability.

It identifies conservation priorities, guides action plans, law and funding, and is updated regularly. In India, the Botanical Survey of India and the Zoological Survey of India compile Red Data Books of threatened plants and animals. Examples: Nepenthes khasiana, Endangered; Santalum album, Vulnerable. The limitation is that it is a list and not a legal instrument; protection needs law, such as the Wildlife (Protection) Act, 1972 and the Biological Diversity Act, 2002.