RBC Metabolism (Part 2) : Mechanisms of preventing oxidative denaturation of hemoglobin

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  Fig. Enzyme that converts MetHb to Hb

  Known mechanisms of preventing or reversing oxidative denaturation of hemoglobin in the erythrocyte include 
• the methemoglobin reductases
• superoxide dismutase
• glutathione peroxidase
• catalase.

(Fig. in left NADH_cytochrome_B5_reductase)

Methemoglobin Reduction

• Most methemoglobin in the erythrocyte is reduced through the action of an enzyme cytochrome b5 methemoglobin reductase, which acts in the presence of two electrons carriers, cytochrome b5, and NADH.
• Only a small amount of methemoglobin is reduced by all other pathways of methemoglobin reduction together. These other pathways involve two that cause the reduction of methemoglobin non-enzymatically, ascorbic acid and glutathione, as well as a second enzyme, NADPH-flavin reductase.
• Deficiency of cytochrome b5 reductase, but not of NADPH-flavin reductase, is associated with methemoglobinemia, confirming that cytochrome b5 reductase is the most important physiologic means of reducing methemoglobin. In vitro evidence also confirms that cytochrome b5 is the rate-limiting factor in methemoglobin reduction.
• Cytochrome b5 reductase has been referred to by several other names, including diaphorase I, DPNH dehydrogenase I, NADH dehydrogenase, NADH methemoglobin reductase and NADH methemoglobin-ferrocyanide reductase. 
• Early work by Gibson in the 1940s demonstrated a relationship between the reduction of methemoglobin and the metabolism of lactate to pyruvate, thus implying an important role for NADH. 
• Eventually, two methemoglobin-reducing enzymes were isolated. 
• The NADH-dependent enzyme, which was absent from several patients with methemoglobinemia, has been shown to be a flavoprotein, with one mole of flavinadenine dinucleotide (FAD) per mole of apoenzyme. Its molecular weight is approximately 34,000. Several investigators have identified the corresponding cDNA, and the gene has been localized to chromosome 22. Most likely, erythrocyte cytochrome compounds b5 reductase and hepatic cytochrome b5 reductase are the product of a single gene.
• The reduction of methemoglobin by highly purified cytochrome b5 reductase in the presence of NADH is extremely slow, implying that another factor is most likely required as an electron carrier. 
• In vitro, this role can be filled by dyes or by ferrocyanide. 
• In vivo, cytochrome b5 acts as the intermediate electron carrier. 
• Erythrocyte cytochrome b5 greatly accelerates reduction of methemoglobin by cytochrome b5 reductase and can also serve as a substrate for hepatic microsomal cytochrome b5 reductase.
• Congenital methemoglobinemia resulting from a deficiency in cytochrome b5 has been described.

Proportion of Methemoglobin Reduction by Various Erythrocyte Systems
Cytochrome b5 reductase	67%
Ascorbic acid	16%
Glutathione	12%
NADPH methemoglobin reductase	5%
Total	100%

• The process by which cytochrome b5 reductase and cytochrome b5 reduce hemoglobin in the presence of NADH probably involves three steps. 
• In the first, NADH binds to the FAD-reductase complex and, in the presence of hydrogen ion, the NAD is converted to NAD+, and the FAD becomes FADH2. 
• In the second step, cytochrome b5-Fe3+ is reduced to cytochrome b5-Fe2+, and the FADH2 reverts to FAD. 
• Finally, methemoglobin forms a bimolecular complex with reduced cytochrome b5 through electrostatic interactions between negatively charged groups around the cytochrome heme and positively charged groups around the heme moieties of methemoglobin.
• The reduction of methemoglobin then takes place and can be represented as follows:
• HbFe³⁺ + Cytb₅Fe²⁺-------->HbFe²⁺ + Cytb₅Fe³⁺
• Of lesser physiologic importance is the enzyme system that depends on NADPH for its activity. It probably accounts for only about 5% of the methemoglobin-reducing activity of normal red cells, and its hereditary deficiency does not lead to methemoglobinemia. The lack of physiologic activity may result from the absence of an intermediate electron carrier analogous to cytochrome b5. 
• If methylene blue is supplied as the carrier, however, the NADPH-dependent enzyme becomes highly effective in methemoglobin reduction. 
• This property is used in the therapy of methemoglobinemia from various causes.

Enzymes Reacting with Products of the Reduction of Oxygen

• As molecular oxygen undergoes successive univalent reductions, a variety of reactive species are generated. These species constitute the oxidizing agents most likely to be responsible for the oxidative denaturation of hemoglobin, and they may damage other cellular components as well, especially lipid-containing elements such as the cell membrane. A variety of mechanisms have evolved in respiring organisms to deal with these potential toxins, and some are found within the erythrocyte.
• Superoxide anions are produced in biologic tissues from several sources, including oxyhemoglobin itself, as well as oxidative reactions catalyzed by flavin enzymes, such as xanthine oxidase. In addition, many drugs and toxins have oxidant activity and appear to generate superoxide. 
• Once superoxide has been generated in aqueous solution, additional toxic products of oxygen may form spontaneously. Thus, superoxide can undergo spontaneous dismutation, yielding peroxide and oxygen:
• O₂^- + O₂^- + 2H⁺--------->H₂O₂ + O₂

• In addition, in the presence of catalytic quantities of transition metals, superoxide and peroxide may react to form the highly reactive hydroxyl radical

• O₂^- + H₂O₂--------> OH^. + OH^- + O₂

• Any of these oxygen derivatives may exert toxic effects on cellular components. 

• As previously noted, superoxide appears to induce methemoglobin formation. 

• It may also bring about cell lysis via its effect on membranes. 

• Hydrogen peroxide is the most stable intermediate in the reduction of oxygen. Although hydrogen peroxide has often been shown to induce the oxidative denaturation of hemoglobin in vitro, whether it does so directly or by giving rise to other products, such as the hydroxyl radical, is not clear.

• The hydroxyl radical is one of the most potent redox agents known. 

• Because it is generated by the radiolysis of water, it is thought to account for many of the effect of radiation in biologic tissue. 

• It also, however, may be generated from superoxide and peroxide and from peroxide in the presence of certain metals:
• Fe⁺² + H₂O₂-------> Fe³⁺ + OH^- + OH^.
• The superoxide dismutases are enzymes that catalyze the dismutation of superoxide to oxygen and peroxide. Although this reaction occurs spontaneously, the presence of this enzyme speeds the reaction to a rate as much as 109 times faster than the spontaneous rate. 
• In the erythrocyte, superoxide dismutase is a soluble, cuprozinc enzyme with a molecular weight of about 32,000. The enzyme accounts for most of the copper content of the red cell, and before its enzymatic function was determined, it was called erythrocuprein or hemocuprein.
• The primary structure of human copper-zinc (Cu-Zn) superoxide dismutase has been determined, and the gene has been mapped to chromosome 21.
• Although superoxide dismutase prevents the formation of methemoglobin in vitro under conditions in which superoxide forms, the relative importance of this reaction in vivo remains to be established.
• Once hydrogen peroxide is formed, two enzymes catalyze the decomposition of hydrogen peroxide is in erythrocytes. The most important of these enzymes is glutathione (GSH) peroxidase, which is a component of the following reaction:

                                                   GSH Peroxidase

                         H₂O₂ + 2 GSH--------------------------->  2 H₂O + GSSG

• The enzyme is effective at very low concentrations of peroxide (Km = 1 X 10^-6M).
• Glutathione peroxidase is the major human selenoprotein, which usually accounts for the anti-oxidant properties of selenium as a micronutrient. Human cells grown in the absence of selenium express significantly reduced glutathione peroxidase activity, despite normal glutathione peroxidase mRNA and transcription levels. The gene for glutathione peroxidase appears to be on chromosome 3, although two homologous genes also appear present in the human genome. 
• The importance of glutathione peroxidase is exemplified by that fact that a genetic defect in the enzyme may lead to a drug-sensitive hemolytic anemia.

• Catalase, the heme enzyme, decomposes hydrogen peroxide to water and molecular oxygen. It appears to be less important to red cell than peroxidase, presumably because it is effective only when the peroxide concentration is relatively high.

• Individuals with hereditary acatalasemia do no develop methemoglobinemia or hemolytic disease; an increase in glutathione peroxidase levels may compensate in part for the lack of catalase.

• Some evidence suggests, however, that erythrocyte catalase may be important in preventing oxidant damage to somatic tissues. Also, the level of catalase increases with physical conditioning, suggesting a physiologically significant role for erythrocyte catalase.

• Catalase consists of a tetramer composed of 60,000 dalton subunits, with four heme groups per tetramer. It is encoded by a gene on chromosome 11. 
• Catalase is a major component of erythrocyte band 4.5 seen of Coomassie-stained gels of erythroid membrane proteins, as the enzyme interacts with the membrane in a calcium and pH dependent manner. 
• Catalase also comprises a major reservoir of erythrocyte protein-bound NADPH. 
• Each tetrameric molecule of erythrocyte catalase contains four molecules of tightly bound NADPH. Although not essential for enzymatic conversion of peroxide to oxygen, the NADPH appears to protect catalase from inactivation by peroxide.
  
  

  Glutathione Metabolism
• Glutathione is the principal reducing agent in erythrocytes and the essential cofactor in the glutathione peroxidase reaction.
• Reduced glutathione (GSH) is a tripeptide (gamma-glutamyl-cysteinyl-glycine). 
   
• Two ATP-dependent enzymatic reactions are required for the de novo synthesis of glutathione:

• glutamic acid + cysteine ------------> gamma-glutamyl-cysteine 
• gamma-glutamyl-cysteine + glycine --------->GSH

• Reaction 1 is catalyzed by glutamyl-cysteine synthetase, reaction 2 by glutathione synthetase. Both reactions can take place in normal erythrocytes. The capacity of normal red cells to synthesize glutathione exceeds the rate of turnover by 150 fold.
  
  
  
  
  
  
  

• In the course of reactions protecting hemoglobin from oxidation, GSH is oxidized, forming oxidized glutathione (GSSG), which consists of two GSH molecules joined by a disulfide linkage, and mixed disulfides with hemoglobin. GSSG rapidly leaves the erythrocyte.
• Thus, maintaining a continuous supply of GSH requires a system to reduce the oxidized forms of glutathione. Such a system is provided by glutathione reductase, which catalyzes the reduction of GSSG by NADPH, a product of the pentose phosphate pathway (Fig 5.27).
• Glutathione reductase also catalyzes the reduction of hemoglobin-glutathione disulfides, yielding GSH and hemoglobin.
  
  
  (Source:  Wintrobe's Textbook of Hematology; Tietz's Textbook of Clinical Chemistry)