Biochemistry: The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

How electrons encounter oxygen, how ATP is generated in the process, and related phenomena.
Hemoproteins
Oxygen-transporting proteins

In Chapter 4, Section D, 5, we discussed the remarkable ability of erythrocyte Hemoglobin to cooperatively bind four molecules of O₂, as well as the structural relationship between hemoglobin and monomeric Muscle Myoglobin, which facilitates the diffusion of O₂ into Tissues [5] and likely serves to store oxygen. Iron in hemoglobin and myoglobin is always in the ferrous state. Erythrocytes possess a specialized system to reduce iron should it accidentally transition to the ferric state (see Chapter 4, Section D, 7, and also Appendix 10-A). According to the most widely accepted view, when O₂ binds to the heme iron, there is no temporary change in the oxidation state of the metal. However, other views exist on this matter [6]. According to Ingram, metal oxidation does not occur due to the aforementioned difficulties in adding a single electron to an oxygen molecule. At the same time, The transfer of two electrons from the metal to oxygen is hindered because the Fe(IV) state is unstable.

It is generally believed that the binding of heme iron to oxygen occurs via the transfer of an electron pair from oxygen to the metal. In unoxygenated hemoglobin, the ferrous ion is in a "high-spin" state; four of the five 3d orbitals of the iron ion valence shell each contain one unpaired electron. The binding of oxygen converts iron into a "low-spin" state, in which all electrons are paired. It is precisely this loss of paramagnetism by hemoglobin upon oxygenation that has long attracted the attention of chemists. According to current concepts, the stability of heme-oxygen complexes is enhanced by an "induced double bond," formed through the shifting of an electron pair from one of the iron atom d orbitals to form a pi-bond with the nearest oxygen atom. Symbolically, this can be depicted as follows:

From these structures, based on the assumption that the initial bond with the metal is formed by a lone electron pair of the O₂ molecule, the most probable bent geometry of the resulting Structure follows, which has recently been unequivocally confirmed for one of the model complexes [8]:

The idea has also been put forward that one of the pairs of oxygen pi-electrons takes part in The formation of the initial bond [4]:

However, the actually observed geometry seemingly rules out the existence of such a structure.

In all oxygen-transporting Hemoproteins, one of the coordination positions of iron, located on the side opposite to the oxygen-binding site, is occupied by the imidazole group of a Histidine side chain. In the absence of such a group, heme cannot bind oxygen; as is known, coordination with heterocyclic nitrogen-containing compounds is favorable for the formation of low-spin iron complexes. Several very simple compounds have been prepared in which a chain bearing an imidazole group at the end is attached to one of the heme rings; it turned out that at an appropriate chain length, these compounds quite successfully reproduce the ability of myoglobin to bind oxygen [7, 8].

Similar compounds having a pyridine ring in the fifth coordination position exhibit a low affinity for oxygen. Thus, the polarizable imidazole group also appears to play a crucial role by facilitating oxygen binding. It has been suggested that the pi-electrons of the imidazole group participate in binding to iron [Equation (10-2)] [7]. The system of pi-bonds encompassing the iron atom enables it to form a stronger bond with the oxygen atom occupying the sixth coordination position.

The scheme presented in Equation (10-2) illustrates another property frequently observed in hemoproteins. The imidazole N—H group is hydrogen-bonded to the C=O group of the peptide chain backbone. Furthermore, it is not difficult to envision a chain of changes triggered by oxygenation, including A change in THE POSITION OF the proton involved in the aforementioned Hydrogen bond and a subsequent charge redistribution within the protein hydrogen-bonding network¹⁾.

An exceptionally interesting discovery was made, namely, that the coordination of heme iron with histidine apparently underlies the cooperativity of hemoglobin binding to oxygen [9, 10]. The radius of high-spin iron in both the ferric and ferrous states is so large that the iron does not fit into the center of the porphyrin ring and is displaced toward the coordinatively linked imidazole group by a distance of 0.06 nm for Fe (II). Thus, in deoxyhemoglobin, both the iron and the imidazole group are located further from the ring plane than in oxyhemoglobin. In the latter, iron resides at the center of the porphyrin ring, since the transition to the low-spin state is accompanied by a decrease in ionic radius [9, 11]. The protein conformation change induced by a small Displacement of the iron ion has already been described (Chapter 4, Section D, 5). However, the true Nature of the "trigger mechanism" leading to these changes is not yet entirely clear. To a first approximation, these changes can be viewed as purely mechanical consequences of shortening the distance between the porphyrin ring and the F-helix of hemoglobin (Fig. 4-17). It has been suggested that this shortening causes a tilt of the heme, which weakens certain Hydrogen Bonds in the Cell/13.html">Protein Structure and ultimately results in the conformational changes already described (Chapter 4, Section D, 5) [10]. Admittedly, this perspective gives insufficient attention to the changes in electron distribution discussed in the previous paragraph. In any case, it is remarkable how effectively nature has taken advantage of minor differences in iron properties induced by a change in the d-orbital electron distribution of this transition metal. One should keep in mind the possibility that transition Metal Ions may induce similar Conformational Changes in other biochemical structures as well.

¹⁾ The carbonyl group shown in Equation (10-2) is part of the F-helix in Hemoglobins and is hydrogen-bonded to other amide groups. The withdrawal of an electron into the heme-oxygen complex will lead to the strengthening of the hydrogen bond shown in Equation (10-2) and to the weakening of the competing intrahelical hydrogen bond. The consequence of this will be a charge redistribution in the upper part of the F-helix, promoting conformational changes followed by structural rearrangement, as shown in Fig. 4-19. Directly adjacent to the F- and G-helices is the beta,a₂ contact, in the region of which Changes in the hydrogen-bonding system take place.

The oxygen carrier in several invertebrate groups, such as the peanut worms Sipunculidoidea, is a non-heme iron-containing protein, hemerythrin [11a]. The subunits of this protein, containing about 113 amino acid residues, frequently form octamers with C₄ Symmetry. Each monomer possesses an active center containing two Fe(II) atoms separated by a distance of 0.34 nm. It is believed that the oxygen molecule is situated between the iron atoms, as indicated in the scheme below (taken from Klotz et al. [11a]):

It is assumed that the O₂ molecule accepts two electrons, oxidizing both iron atoms to the Fe(III) state while converting itself into the peroxide dianion O^2-₃. The process is fully reversible. The transformation of oxygen into a bound peroxide ion is confirmed by Resonance Raman scattering studies (see Chapter 13, Section B, 3,6).

Similarly, the blue copper-containing hemocyanin of many invertebrates binds one O₂ molecule per two Cu(I) atoms. Oxygen presumably forms a bridge between the two copper atoms. Since CO does not bind to this protein (unlike hemoglobin iron), Ingram [4] proposed a non-linear configuration for the complex. The oxygenated compound has a bright blue color and absorbs light 5–10 times more strongly than other known univalent copper complexes. Based on this, it is believed that some of the copper atoms may change their oxidation state upon oxygen binding. Further support for this idea came from the observation that treating oxygenated hemocyanin with glacial acetic acid yields equivalent amounts of cuprous and cupric ions:

(CuО₂Cu)²⁺+ Н+ → Cu²⁺ + Сu⁺ + НO₂.      (10-3)

Another product is thought to be the hydroperoxide radical. Hemocyanins are large oligomeric molecules that present a very striking appearance under the Electron microscope [11b, 11c].

Many iron- and copper-containing Proteins bind O₂ in a manner analogous to hemoglobin, myoglobin, and hemocyanin, but subsequently the oxygen is "activated" and participates in further reactions. We will examine these Enzymes later, but first let us consider the group of heme-containing enzymes that function as electron carriers.

Supplement 10-A

Glutathione Peroxidase and Erythrocyte Abnormalities

The processes by which hemoglobin is maintained in the Fe(II) state and Functions normally in intact erythrocytes are critical to our health. Numerous inherited disorders leading to anemia have helped to elucidate the outlined biochemical pathway^a.

About 90% of the glucose utilized by erythrocytes is converted to lactate via Glycolysis, but ~10% is oxidized (via glucose 6-phosphate formation) to 6-phosphogluconate. This oxidation (reaction a) is catalyzed by glucose-6-phosphate dehydrogenase [Eq. (8-42)] with the participation of NADP⁺. This specific reaction primarily supplies erythrocytes with the necessary amount of NADPH used to reduce glutathione (Supplement 7-G) in reaction b. Glucose-6-phosphate dehydrogenase is of paramount importance; yet over 100 million people, particularly in tropical and Mediterranean countries, have an inherited deficiency of this enzyme. As it turns out, these Genetic Disorders are quite heterogeneous—at least 22 types of such abnormalities have already been discovered. It has been established that the absence of this enzyme leads to very serious consequences: in certain diseases, as well as in response to the administration of specific drugs, the destruction of A large number of erythrocytes is observed. The survival of defective genes, as in the case of Sickle-Cell Anemia (Supplement 4-D), appears to be due to the enhanced resistance to malaria of individuals carrying these genes.

Other erythrocyte defects causing hypersensitivity to drugs are associated with a deficiency of glutathione (due to a reduced rate of its synthesis) or glutathione reductase (reaction b). As it turned out, in these cases, the disorders caused by drug administration result from the formation of H₂О₂ (reaction d). According to current concepts, the function of glutathione and the enzymes catalyzing reactions a, b, and c is to destroy the hydrogen peroxide generated either through metabolic reactions or via the autooxidation of drugs. In human erythrocytes, the principal enzyme destroying H₂О₂ (reaction c) is a selenium-containing peroxidase (Supplement 9-E); catalase likely performs the same function [Eq. (10-5)], and both enzymes appear to be required for normal viability of the Organism.

Excess H₂О₂ damages erythrocytes in two ways. First, it causes excessively vigorous oxidation of functional hemoglobin to Fe(III)-containing methemoglobin (methemoglobin is also formed spontaneously during The transport of oxygen by hemoglobin; it is estimated that up to 3% of hemoglobin may be oxidized to methemoglobin per day). The resulting methemoglobin is reduced back to hemoglobin by NADH-methemoglobin reductase (reaction e). A smaller fraction of methemoglobin is reduced by a similar NADPH-dependent enzyme (as indicated by the arrow leading from NADH to reaction e). Cases of inherited NADH-methemoglobin reductase deficiency are known.

The second damaging function of H₂О₂ is the attack on the double bonds of Unsaturated Fatty acids in cell membrane Phospholipids. The resulting fatty acid hydroperoxides can trigger further reactions leading to C—C bond Cleavage and membrane disruption. This is considered the primary cause of hemolytic anemia that develops in certain patients upon drug administration. Glutathione peroxidase most likely also destroys fatty acid peroxides. Maintaining erythrocytes in a normal state also requires vitamin E, which exhibits antioxidant activity (Supplement 10-B).

Some cases of granulomatosis (Section B, 6) are accompanied by a decrease in glutathione peroxidase activity as well as a reduction in the microbicidal activity of phagocytes. It is hypothesized that fatty acid hydroperoxides impair normal phagocytosis by inhibiting certain enzymes^b.

^a Chanarin I. In: Biochemical Disorders in Human Disease, 3rd ed. (R. H. S. Thompson and I. D. P. Wooton, eds.), pp. 163–173, Academic Press, New York, 1970.

^b Serfass R. E., Ganther H. E., Nature (London), 255, 640—641 (1975).

Supplement 10-B

Vanadium

The possible biochemical function of vanadium is indicated by the presence of vanadocytes, green Blood Cells containing 4% V(III) and 1.5—2 N H₂SO₄. These cells were discovered in tunicates (marine sea squirts, Ch. 1, Sec. D,1)^a. It has been suggested that the V-containing protein vanadochrome acts as an oxygen carrier. However, there is as yet no definitive proof of this, and the function of the protein remains unclear. Vanadium is accumulated by several other marine organisms and is present in animal tissues at a level of ~0.1 ppm.

It has recently been demonstrated that rats have an essential dietary requirement for vanadium^b,c. It is likely also essential for humans, who typically ingest ~2 mg of vanadium per day. The adult human body contains ~30 mg of vanadium. A possible function is related to Lipid METABOLISM. Vanadium can exist in multiple oxidation states, ranging from +2 to +5. In alkaline solution, vanadium is present predominantly as the VO^3-₄ ion, containing pentavalent vanadium. The VO²⁺ vanadium ion, which features a double bond, is a particularly stable form of V(IV). The chemical properties of vanadium suggest that it may participate in oxidation-reduction processes.

Interest in vanadium as an essential dietary component was stimulated by observations showing that high doses of vanadium inhibit Cholesterol synthesis and lower blood levels of phospholipids and cholesterol. Vanadium has also been reported to help prevent dental caries by promoting tooth mineralization. Unlike tungsten (Supplement 14-A), vanadium does not exhibit competition with molybdenum in the animal organism^d. However, an inactive vanadium-containing Nitrogenase has been found in Azotobacter.

^a Carlisle D. B., Proc. Roy. Soc., B, 171, 31—42 (1968).

^b Schwarz K., Milne D. B., Science, 174, 426—428 (1971).

^c Hopkins L. L., Jr. In: Trace Element Metabolism in Animals (W. A. Hoekstra, J. W. Suttie, H. E. Ganther and W. Mertz, eds.), Vol. 2, pp. 397—406, University Park Press, Baltimore, 1974.

^d Johnson J. L., Rajagopalan K. V., Cohen H. J., JBC, 249, 859—866 (1974).