Metalloenzymes

Redox and non-redox metalloenzymes - Introduction and examples , Copper blue proteins - Classifications and examples, structure and mechanistic action of ascorbic acid oxidase; Peroxide and superoxide scavenger enzymes: Structure and Reactivity of superoxide dismutase, catalase and peroxidase

1. BIO-INORGANIC CHEMISTRY-III II CA - WORKSHEET NAME : MOHAMMED FAYAZ FURKHAN REG NO : 2201712049022 CLASS/YR : II MSc Chemistry

2. REDOX METALLOENZYMES: Definition: Redox metalloenzymes are a class of enzymes that contain metal ions in their active sites and participate in electron transfer reactions. Role of Metal Ions: Metal ions act as cofactors in redox metalloenzymes , facilitating electron transfer between the substrate and the enzyme. Electron Transfer Reactions: Redox metalloenzymes catalyze redox reactions, where one substrate is oxidized, and another is reduced. The metal ion undergoes changes in oxidation state during the reaction, facilitating electron transfer. Examples: Cytochrome c oxidase, a redox metalloenzyme found in the mitochondria, transfers electrons from cytochrome c to oxygen during cellular respiration. Ferredoxin , another redox metalloenzyme , transfers electrons in photosynthesis and other metabolic processes. Coordination Environment: The metal ion in redox metalloenzymes typically resides in a specific coordination environment provided by surrounding amino acid residues.

3. NON-REDOX METALLOENZYMES Definition: Non-redox metalloenzymes are enzymes that contain metal ions in their active sites but do not participate in direct electron transfer reactions. Role of Metal Ions: Metal ions in non-redox metalloenzymes serve various functions, such as providing structural stability or acting as Lewis acids/bases for catalysis. Catalysis: Non-redox metalloenzymes use metal ions to promote substrate binding and assist in catalytic reactions without undergoing changes in their oxidation states. Examples: Carbonic anhydrase is a non-redox metalloenzyme that contains zinc ions and catalyzes the hydration of carbon dioxide to bicarbonate. Ribonucleotide reductase contains a stable radical in its active site and uses a manganese center to generate the radical required for catalysis. Coordination Environment: Similar to redox metalloenzymes , non-redox metalloenzymes also have specific coordination environments around the metal ions, which are critical for their functions.

4. In summary, redox metalloenzymes participate in electron transfer reactions, utilizing metal ions in their active sites to facilitate redox reactions. On the other hand, non-redox metalloenzymes do not undergo direct electron transfer but utilize metal ions for various functions, such as structural stability and catalysis, without changes in oxidation states. Both types of metalloenzymes play crucial roles in numerous biological processes.

5. Cu-proteins have been classified into Type 1, Type 2, and Type 3 Cu-sites based on their structural and spectroscopic properties. Blue copper proteins exhibit an intense charge transfer band around 600 nm, attributed to Type 1 Cu centers . Multicopper blue proteins contain multiple types of Cu centers (Type 1, Type 2, Type 3) and demonstrate oxidase activity. Proteins with a single Type 1 Cu center primarily function as electron-transfer proteins. Nonblue copper proteins, such as galactose oxidase, cytochrome c oxidase, and superoxide dismutase, also exist. Examples: Copper blue proteins – classification and examples

6. Structure and Mechanistic activity of Ascorbic Acid Oxidase : A Blue Oxidase Protein Blue Protein: Found in plants and microorganisms. Functions as ascorbic acid oxidase (AAO). AAO Enzyme: Catalyzes the oxidation of ascorbic acid (preferably L-form) to dehydroascorbic acid. Uses O₂ as a reactant, reducing it to H₂O. Cu²+ ions can also catalyze ascorbic acid oxidation, producing H₂O₂. Enzyme Structure: AAO has two subunits, each with a molecular weight of 75 kDa . Each subunit consists of two polypeptide chains. AAO contains a total of 8 copper centers per molecule .

7. Cu-Sites in AAO: Each subunit has four copper centers : One Type 1 Cu center . One Type 2 Cu center . Two Type 3 Cu centers . Laccase : Another multicopper oxidase with a similar Cu-site structural position as AAO. The oxidized form of AAO exhibits an intense blue color due to the Type 1 Cu site, while the reduced form is colorless . Oxygen Reactivity: O₂ reduced to H₂O by AAO during ascorbic acid oxidation. Cu²+ ions alone catalyze ascorbic acid oxidation, producing toxic H₂O₂.

8. Electron Transfer Pathway: Electrons from the organic substrate (ascorbic acid) enter the protein through a histidyl residue ligated to Type I Cu (blue copper center ). The electrons are transported through a series of covalent bonds to reach the trinuclear site, which consists of Type 2 and Type 3 Cu centers located about 13 Å away. At the trinuclear site, O₂ ( dioxygen ) is reduced to H₂O. Dioxygen Binding: Dioxygen binding occurs within the trinuclear array of Cu-sites. Rapid spin exchange takes place among the Cu-sites during the process. Electron Transport and Efficiency: Electron transport is mediated through covalent bonds in the protein, enabling efficient electron transfer. The arrangement of catalytic and electron transfer sites optimizes the efficiency of electron transfer. AAO Enzyme Mechanism: AAO oxidizes ascorbic acid in two successive one-electron transfer steps, forming a free radical intermediate. The free radical is further oxidized to produce the final product. The active form of the enzyme is regenerated from the reduced form by O₂. The reduced form of the multicopper protein reduces O₂ to H₂O through a four-electron transfer process.

9. Ascorbate Radical Reactions: Enzymatic and nonenzymatic reactions involving the ascorbate radical. Nonenzymatic reaction produces dehydroascorbic acid and ascorbic acid molecules. AAO- Catalyzed Reaction: AAO reaction rate is independent of O₂ concentration. Cu²+- catalyzed reaction in vitro shows a first-order dependence on O₂ concentration. Cu(II)-Chelate as Model Compound: Cu( trien )2+ can act as a model compound for AAO. Activity of AAO: Activity of AAO increases during infection. AAO Enzyme Inhibition: AAO may be inhibited by various compounds like sulfide , xanthate , diethyldithiocarbamate , oxine , and cyanide.

10. SUPEROXIDE DISMUTASE (SOD) Toxic Substances from Partial O₂ Reduction: Partial reduction of O₂ may generate toxic substances like superoxide (O₂₂) and peroxide (O₂). Some enzymes, like cytochrome c oxidase, can convert O₂ to H₂O (4e reduction) without forming toxic intermediates . Disproportionation Reactions and SOD Enzymes: Efficient mechanisms needed to prevent O₂ and H₂O₂ accumulation in living systems. Catalase catalyzes the disproportionation of O₂₂, while superoxide dismutase (SOD) catalyzes the disproportionation of O₂. Three types of SODs: Cu, Zn-SOD (eukaryotic cells), Fe-SOD, and Mn -SOD (prokaryotes). Cu, Zn-SOD is stable and manages hazards from O₂ effectively.

11. Disproportionation Mechanism: Superoxide disproportionation occurs through a ping-pong type mechanism in two one-electron transfer steps. Aqueous Cu(II) ion catalyzes the reaction about 104 times faster than the uncatalyzed reaction at pH = 7.0. SOD Enzymatic Disproportionation Rates: Cu, Zn-SOD: 2 x 10⁹ M-1 s-1 Mn -SOD: 5.5 x 10⁷ M-1 s-1 Fe-SOD: 3 x 10⁸ M-1 s-1 Dangers of Excessive O₂ Accumulation: Excessive O₂ accumulation can lead to oxidative stress-induced diseases. O₂- can produce OH radicals, causing various hazards . Synthetic Enzymes ( Synzymes ): Attempts to prepare low molecular weight synthetic enzymes ( synzymes ) as therapeutic agents mimicking natural SOD activity.

12. Bovine Erythrocyte Superoxide Dismutase (BSOD): Dimeric enzyme with a molecular weight of 32 kDa . Represents as Cu₂Zn-BSOD with two subunits, each containing one Cu site and one Zn site at the active site. Subunits are bridged through a deprotonated imidazole ring of a histidyl chain (His-61). Cu(II) and Zn(II) Coordination: In resting condition, Cu(II) is in a square planar geometry. Cu(II) is equatorially coordinated to four histidyl imidazoles (His-46, His-118, His-44, and His-61) and weakly bound to a H₂O molecule axially. Zn(II) is in a tetrahedral geometry, coordinated to three histidyl imidazoles (His-78, His-69, and bridging His-61) and the carboxylate group of Asp-81.

13. Role of Zn(II) in Cu, Zn-SOD: Zn(II) center has a primarily structural role. Zn- center is buried in the protein, and removal of Zn(II) does not significantly affect SOD activity. Replacing Zn(II) with other metal ions does not change SOD activity. Cu(II) presence is essential for SOD activity . Zn-Depleted Protein: Zn-depleted protein lacks thermal stability and denatures at a lower temperature than the native protein. Supports the notion that Zn(II) primarily plays a structural role in the enzyme . These key points summarize the characteristics of Bovine Erythrocyte Superoxide Dismutase (BSOD), the coordination of Cu(II) and Zn(II) in the enzyme's active site, and the structural role of Zn(II) in Cu, Zn-SOD, which is essential for its stability and thermal denaturation.

14. Structural Features of Catalase and Peroxidase Horseradish Peroxidase (HRP): HRP is extensively studied among peroxidases. X-ray studies of HRP and beef-liver catalase indicate high spin Fe(III) in the heme b group in their resting state .

15. Catalase Structure and Active Site: Catalase has four Fe(III)- heme units in separate protein environments with a total molecular weight of 250 kDa . In the active site cavity of catalase, one axial position is occupied by a phenolate group from a tyrosyl residue, and the other is occupied by a water molecule, which is replaced by H₂O₂ during catalysis . Peroxidase Structure and Active Site: HRP, in contrast, has a single Fe(III)- heme unit with a molecular weight of 40 kDa . At the active site cavity of peroxidase, one axial position is coordinated by an imidazole moiety of a histidyl residue, and the second axial position is reserved for peroxide binding. Histidine and aspartate or asparagine side chains at the active site of peroxidase are suitably oriented to interact with the bound hydrogen peroxide during the catalytic reaction . Catalytic Reaction : In both catalase and peroxidase, the catalytic reaction involves the heterolytic cleavage of the O-O bond of hydrogen peroxide, which involves charge separation.

16. Reactivity : Catalase: Catalase has a high turnover number, meaning it can rapidly process large amounts of H₂O₂. It primarily functions to decompose H₂O₂ into water (H₂O) and oxygen (O₂), preventing the accumulation of toxic H₂O₂. Catalase has a broad pH range for optimal activity, typically around pH 7 . Peroxidase: Peroxidase has a lower turnover number compared to catalase. It can catalyze the breakdown of H₂O₂ as well, but it also has broader substrate specificity. Peroxidases can use H₂O₂ to oxidize various substrates, including phenols and other organic compounds. The optimal pH for peroxidase activity varies depending on the specific peroxidase type.