Metals in Biology – Exam Review Notes

Methane‐Oxidizing Bacteria, Hot Springs & Environmental Relevance

• Thermophilic springs (e.g. Bath, England) harbor Methylococcus sp. → convert atmospheric $CH<em>4$ → $CH</em>3OH$
• Tourism/“healing” waters: bacterial bio-film removes skin irritants; compared to “holy water.”
• Bioremediation: Exxon Valdez (1989), Prince William Sound – inoculation with “oil-eating” Methylococcus restored coastline; MMO enzymes degrade long-chain hydrocarbons.

Soluble Methane Mono-Oxygenase (sMMO)

• Protein complex: reductase + regulatory + hydroxylase (MMOH).
• Active core (MMOH) = di-iron center bridged by O/Carboxylates:
• Fe1: 2 Glu + 1 His
• Fe2: 1 Glu + 1 His + H$_2$O
• Resting state: $Fe^{II}–Fe^{II}$.
• Crystal shows unsym. di-iron but cooperative redox.

Catalytic Cycle (canonical mechanism)

1. $ext{MMOH}<em>{red}\, (Fe^{II}Fe^{II}) + O</em>2 \xrightarrow[]{+ e^-} (Fe^{III}Fe^{II})\, -\, O_2^{!<em>}$ superoxo (P$^$).

2. Second e$^-$ → $Fe^{III}Fe^{III}\,(\mu! ext{‐}O<em>2^{2-})$ (P) – λ${max}$ 720 nm.

3. $O–O$ scission → Q: $Fe^{IV}Fe^{IV}(\mu!‐O)<em>2$ – λ${max}$ 420 nm; EPR silent; Mössbauer $\delta!\approx!0.2$ mm s$^{-1}$.

4. H-atom abstraction + OH rebound: $CH<em>4 \to CH</em>3OH$; cluster returns $Fe^{III}Fe^{III}(\mu!‐OH)_2$, then re-reduced.

Revised Mechanistic Nuances

• Alternative: P hydroxylates substrate directly; or Q forms transient $Fe^{IV}Fe^{IV}(\mu!‐O)(\mu!‐OH)$ (oxo–hydroxo) before rebound.
• Spectroscopically distinct intermediates allow stopped-flow trapping.

Kinetics – Double-Mix Stopped-Flow

• Drive 1: $Fe^{II}Fe^{II} + O<em>2$ (pump 1) → build P or Q (30–500 ms). • Drive 2: inject substrate (pump 2). • Monitor 720 nm (P) or 420 nm (Q). • Pseudo-first-order plot $k</em>{obs}$ vs. [substrate] → slope = $k<em>{cat}/K</em>M$.

Kinetic Isotope Effects (KIE)

• CH$4$ vs. CD$4$: $k<em>H/k</em>D \approx 23 – 26$ → C–H scission rate-limiting; tunnelling indicated.
• C$2$H$6$/C$2$D$6$: KIE ≈ 1 → diffusion/binding, not C–H cleavage, limits rate.
• Class I substrates (high KIE > 7): $CH<em>4, CH</em>3NO<em>2, CH</em>3CN$ – H-atom abstraction RDS.
• Class II (KIE ≈ 1): $C<em>2H</em>6, CH_3OH$ – substrate access RDS.

Radical vs. Concerted Pathways

• DFT (Lippard, Maiti):
• TS1 (C–H break, O–H form) $\Delta G^{\ddagger} \approx 18$ kcal mol$^{-1}$.
• Radical rebound favored: $Fe^{III}Fe^{IV}(\mu!‐O)(\mu!‐OH) + CH<em>3^·$ → $CH</em>3OH$ (low barrier).
• Concerted “hydroxylation/transfer” ~3–5 kcal higher.

Oxygen Evolving Complex (PS II)

• Photosynthetic $H<em>2O \to O</em>2$ at thylakoid lumen.
• Cluster: $Mn<em>4CaO</em>5$ (distorted chair) + ligating Asp/Glu/His; updated XFEL models minimize X-ray damage.

Kok Cycle (light-driven)

$S<em>0 \xrightarrow{h\nu} S</em>1 \xrightarrow{h\nu} S<em>2 \xrightarrow{h\nu} S</em>3 \xrightarrow{h\nu} S<em>4 \xrightarrow{\text{dark}} S</em>0 + O<em>2$ • Each $h\nu$ ⇒ 1 e$^{-}$ removed (multi-step PCET). • Proposed oxidation states: – S$0$: $Mn^{III}<em>2Mn^{IV}</em>2$ or $Mn^{II}Mn^{III}<em>3$ – S$3$: predominantly $Mn^{IV}<em>4$ – S$4$: transient $Mn^{V}Mn^{IV}_3$ oxo ready for O–O coupling.

O–O Bond Formation Models

1. Nuc‐Electro $Ca^{2+}–OH^-$ attacks terminal $Mn^{V}=O$ (oxyl).

2. Bridging $\mu!‐O$ couples with $Mn^{IV}–OH$ (radical oxo–hydroxo).
   • EXAFS: Mn–Mn 2.7 Å contractions in S$_3$; FTIR/EPR multiline support mixed-valent allocations.

Metal Ion Pumps & Channels

Energy-Driven Pumps (Active Transport)

• Na$^+$/K$^+$-ATPase: 3 Na$^+$ out / 2 K$^+$ in per $ATP \to ADP + P_i$; Asp-P intermediate.
• Ca$^{2+}$-ATPase: cytosolic Ca$^{2+}$ extruded via Asp-351-P cycle.

Passive Channels (Facilitated Diffusion)

• K$^+$ channel (KcsA prototype) – selectivity filter TVGYG narrows to 3 Å.
• Selectivity:
– Large ions (Rb$^+$, Cs$^+$) excluded sterically.
– Small ions (Na$^+$, Li$^+$) retain hydration shell; desolvation $\Delta G_{hyd}$ (Na$^+$ −99 kcal mol$^{-1}$) too high vs. coordination to backbone carbonyls; K$^+$ (−80 kcal mol$^{-1}$) optimally balanced.
• Rapid flux: multi-ion knock-on mechanism – electrostatic repulsion accelerates throughput.

Oxygen Transport Proteins

Hemoglobin / Myoglobin (heme Fe)

• $Fe^{II}$ (high-spin) out-of-plane; O$2$ binding → $Fe^{III}–O</em>2^{·-}$ low-spin; resonance Raman \tilde{\/\nu}{O–O}=1105 cm$^{-1}$. • Cooperativity in Hb (sigmoidal $\theta$ vs. $pO</em>2$); single-site binding in Mb.
• Synthetic mimics: picket-fence porphyrin, capped porphyrins – block dimeric \mu$-O/OH formation.

Hemerythrin (di-Fe, non-heme)

• Differential His ligation (3 His + bridges vs. 2 His).
• O$2$ reduction: $Fe^{II}</em>2 \to Fe^{III}<em>2(\mu!‐O</em>2H)(\mu!‐OH)$ (peroxo-hydroxo); Raman \tilde{\/\nu}_{O–O}=844 cm$^{-1}$.
• Synthetic 3N/2N di-iron complexes replicate spectra & reversibility.

Hemocyanin (di-Cu)

• Tris-His $Cu^{I}<em>2$ (colourless) → $Cu^{II}</em>2(\mu!‐\eta^2":[O<em>2]^{2-})$ (blue) \tilde{\/\nu}{O–O}=750 cm$^{-1}$.
• Same core serves in Tyrosinase for ortho-hydroxylation (cresolase/catecholase) when phenol substrate present.

Copper–Dioxygen Reactivity (Synthetic & Enzymatic)

Dinuclear Motifs

• Tetradentate ligands → end-on $Cu^{II}<em>2(\mu!‐1,2‐O</em>2^{2-})$ (nucleophilic).
• Tridentate → side-on $Cu^{II}<em>2(\mu!‐\eta^2:\eta^2‐O</em>2^{2-})$ (electrophilic).
• Bidentate → $Cu^{III}<em>2(\mu!‐O)</em>2$ (bis-oxo).
• Reactivity: phenolate ortho-OH, aliphatic C–H abstraction, O-atom transfer, depending on core.

Mononuclear Copper–O$_2$ Species

• $Cu^{II}(O<em>2^{·-})$ (λ${max}$ 345 nm, EPR $g_{\parallel}!\approx!2.23$) – generated at −80 °C.
• PHM/DBM: superoxo vs. cupryl $Cu^{III}=O$ vs. hydroperoxo debated; all achieve sterically directed H-atom abstraction → R–OH.
• Ascorbate recycles $Cu^{II} \to Cu^{I}$.

Comparative Themes & Key Numbers

• Peroxo λ${max}$: MMO 720 nm, Cu$2$-side-on 340-350 nm.
• Q-state (Fe$^{IV}2$): 420 nm, Mössbauer $\delta=0.18$ mm s$^{-1}; EPR silent. • O–O Raman bands: – 1105 cm$^{-1}$ (Fe–superoxo) – 844 cm$^{-1}$ (Fe$2$-peroxo)
– 750 cm$^{-1}$ (Cu$2$-peroxo) • K${H}/K{D}$: 26 (CH$4$), ≈1 (C$2$H$6$).
• K$^+$ channel pore ~3 Å; Na$^+$ hydration shell radius ~3.6 Å.

Conceptual Take-Aways

• Metal centres enable multi-electron redox with stepwise PCET; protein matrix edits outcome (react vs. transport).
• Isotopic, spectroscopic (UV/Vis, Mössbauer, EPR, Raman), and fast-kinetics techniques disentangle fleeting intermediates.
• Ligand design (in enzymes or synthetic models) dictates nuclearity, O$2$ binding mode, electrophilic/nucleophilic character, and downstream chemistry. • Nature re-uses identical cores (e.g. Cu^{II}2(O_2^{2-})$$) for drastically different purposes by substrate positioning and secondary-sphere tuning.