Haemoglobin and Similar Molecules

Csopak, and B. Lindman, Arch. Biochem. Biophys., 1974, 162, 552. 

Human oxyhaemoglobin (Hb02) in the presence of excess nucleophile ie.g. N, SCN , F , Cl ) has been shown to form cleanly the oxidized methaemoglobin (metHb) with the nucleophile as the ligand. The rates, which are sensitive to pH and the nucleophilicity of the anionic nucleophile (N ), obey the law Rate = [HbOJ-[N ][H+]. This autoxidation process therefore appears to involve the nucleophilic displacement of superoxide from a protonated intermediate and can reasonably account for normal metHb formation in the erythrocyte where Cl can serve as the nucleophile. [MetHb formation by electron-transfer agents such as NOg , which are not normally present, can follow a different course, e.g. direct electron transfer to bound Og to form iron(m) peroxide.] 

Baldwin and Huff have invoked mechanistic ideas in designing an iron(n) complex which should share with haemoglobin the ability to bind oxygen reversibly in solution rather than suffer the irreversible autoxidation to iron(iii) species experienced by all other known complexes of Fe. Studies on the autoxidation mechanism for iron(u) salts have suggested that an initial 1 1 binding of dioxygen by the metal [step a, equation (1)] is followed by a rapid bimolecular redox process 

The kinetics of the rebinding of CO to myoglobin and to cytochrome P450 after removal by a light flash have been reported. Above 240 K the reaction is second order while between 240 and 200 K the rebinding becomes exponential and independent of the CO concentration below 150 K the reaction follows a power law 

) is thought to be dissociative in character, as with most octahedral metal complexes. If the normal la mechanism is operating, it is to be expected that ligand substitution [equation (3)] would involve aquation followed by anation. This Mb-L + Y= Mb-Y + L (3) 

Kinetic studies have been reported on the oxidation of horse and human Hbs by copper(u) and the reduction of Hb and Mb by chromium(ii). 

The structure of horse metHb has been determined at 2.0 A resolution. The increased resolution (c/. 5.5 and 2.8 A) is sufficient to show up a number of bound water molecules in the contact regions between the subunits which had been overlooked previously, but it is not, unfortunately, sufficient to decide whether the porphyrin rings are fiat or slightly domed, puckered or ruffled. The effects of pressure on the visible spectra of metHb and metMb have been reported (together with those of cytochrome c and horse radish peroxidase) the effect is to shift the equilibrium between open and closed crevice structures in favour of the latter. The spectrum of the short-lived intermediate formed in the reaction of eaq with metHb is consistent with a low-spin iron(ii) species the rate of its subsequent transition to the stable high-spin derivative is solvent dependent. The results of a resonance Raman study on inositol hexaphosphate binding to metHb fluoride are consistent 

Beetlestone and co-workers have used the opportunity of reporting the formation constants of the azide complexes of two metHbs to summarize much of their work on the reactivity differences between Hbs. 

The structure of metmanganoglobin, in which Mn i replaces Fe as the haem metal, has been compared with that of native Hb. Kinetic evidence has been presented for an intermediate in the binding of azide to metMnMb it is seen as a weak Mn +-azide complex in which the metal ion remains out-of-plane toward the imidazole of the proximal histidine, the metal lying toward the anion in the final complex. 

The kinetics of the reaction of Mb with CO and Og have been studied as a function of temperature in various mixtures of glycerol and water in order to examine the importance of diffusion on protein-ligand reactions. The apparent values of for the binding reaction vary with temperature in a way which is consistent with a change from chemical activation of the reaction at higher temperatures to diffusion control at lower temperatures and higher viscosities. Linear correlations are found between Aff and A5 for the chemical activation-controlled portions of the reactions, corresponding to isokinetic temperatures of 305 and 288 K for the CO and Oa reactions, respectively. 

A new model, based on the bonding in ozone, has been proposed for the bonding of an O 2 molecule to the iron of Hb and myoglobin (Mb). It involves basically a biradical with a singlet state stabilized by a three-centre four-electron jT-bond and leads to an easy rationalization of the facile formation and dissociation of the Fe—O2 bond since the O2 would always retain its triplet ground state character. 

Reduction of one of the four haem groups of human aquometHb A has been studied by pulse radiolysis. In unbuffered aqueous solution at 23 °C and pH 6.2 (7=0.1 mol I ), the rate constant for the reaction with 4.2 x 10 1 mol s. Similar results were obtained for cyano- and azido-metHb and for deoxyHb (4.7, 4.4, and 4.3 x 10 1 mol s, respectively). From studies on the redox equilibria of fluoro- and azido-metHb it is concluded that the marked co-operativity and pH-dependence of E are not coupled to changes in the ferrihaem spin state. 

The rate constant for the reduction of metMb by the iron(ii) complex of transA, 2-diaminocyclohexane-iVWV iV -tetra-acetate [Fe(cydta) ] at 25 °C is 28 1 mol s S with = 13 kcal mol and = -11 cal mol Both CN and OH inhibit the reduction because of the relatively low reactivity of cyanometMb (Mb+CN ) and the ionized metMb (Mb+OH ) (rate constants=4.0 x 10 and 4.8 1 mol s , respectively). The kinetics of the reverse reaction, namely the oxidation of oxyMb by Fe(cydta) , are consistent with a scheme in which reaction occurs only through the deoxy form (A = 1.45 x 10 1 mol s ). The authors interpret their data in terms of a simple outer-sphere mechanism. 

Pulse radiolysis has been used to measure the rate constant for the reaction of with coenzyme Big it is 3.0 x 10 1 mol s.  

It has been shown that axial base-ligand exchange in alkyl-(base)cobaloximes occurs by a purely dissociative (D) mechanism in chloroform but it was not possible to distinguish between this and an 7d mechanism for the reaction of pyridine with a series of alkylcobaloximes in water. 

---

Figure 2S.7 Haemoglobin (a) The haem group, composed of the planar FIX molecule and iron, and shown here attached to the globin via an inudazole-nitrogen which completes the square pyramidal coordination of the Fe , and (b) myoglobin showing, diagrammadcally, the haem group in a pocket formed by the folded protein. The globin chain is actually in the form of 8 helical sections, labelled A to H, and the haem is situated between the E and F sections. The 4 subunits of haemoglobin are similar.

The difference in properties between the two proteins, whose tertiary structures are very similar (page 67), is due to the superimposed quaternary structure of haemoglobin and the fact that the ease with which any haem group binds O2 is determined by the state of the other three. Starting with deoxyhaemoglobin the first O2 molecule is taken up very slowly, the second and third are taken up more and more readily and the fourth is taken up several hundred times more rapidly than the first hence the sigmoid shape of the curve. 

The principles of ESR spectroscopy are very similar to NMR spectroscopy but the technique gives information about electron delocalizations rather than molecular structure and it enables the study of electron transfer reactions and the formation of paramagnetic intermediates in such reactions. In some situations, information regarding molecular structure can be obtained when suitable prosthetic groups are part of a molecule, e.g. FMN (flavin mononucleotide) in certain enzymes or the haem group in haemoglobin. Sometimes it is possible to attach suitable groups to molecules to enable their reactions to be monitored by ESR techniques. Such spin labels as they are called, are usually nitroxide radicals of the type... 

Pyridoxal 5 -phosphate (a derivative of pyridoxal vitamin Bg) is similar in size and charge to 2,3-DPG. Covalent attachment of pyridoxal 5 -phosphate reduces the oxygen affinity of the haemoglobin molecule. Covalent attachment of benzene isothiocyanates to the amino termini of the four haemoglobin polypeptide chains, also yields derivatives which display lower oxygen affinity. These may prove worthy of clinical investigation. 

Respiratory pigments similar to the vertebrate haemoglobins have also been identified in many invertebrates. These vary from small proteins with two Fe-porphyrin units to large molecules containing up to 190 Fe-porphyrin units. Myoglobin, the 02 storage protein in muscle tissue, is also a small iron-protoporphyrin protein. The crystal structures of this and a number of other porphyrin proteins are now known (Chapter 20.2, Table 11). 

C—O), which is due to its displacing a similarly shaped molecule, oxygen (O—O), from combination with haemoglobin (Douglas, Haldane and Haldane, 1912). 

The problem of the way in which an animal is able to manufacture the special molecule of haemoglobin that it needs is part of the general problem of the manufacture of specific biological substances. For example, a virus molecule in the proper environment (that provided by its host) is able to cause the production of replicas of itself, and the phenomena of heredity depend upon the similar autocatalytic action of molecules of genes present in the chromosomes and also in the cytoplasm of cells. 

---