The Enzymic Incorporation oj Iron into Protoporphyrin 21 1 



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 Schwartz, H. C, Cartwright, G., Smith, E. L. & Wintrobe, M. M. (1959). Blood 14, 

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DISCUSSION 



The Formation of Metal-Porphyrin Complexes 



Co-ordination of Divalent Metal Ions with Porphyrin Derivatives 

 Related to Cytochrome c 



By J. B. Neilands (Berkeley) 



Neilands: Although the iron bound to the primary, all-oxygen llgands is eventually 

 passed on to the iron-containing enzymes, the mechanism of this transfer and the 

 number of stages involved are still largely unknown. In those instances in which a 

 porphyrin-protein serves as the ultimate repository for the iron, an enzymic incor- 

 poration of the metal ion into the prosthetic group has been indicated (Neve, p. 207). 

 For a more complete understanding of the total biosynthesis of metalloporphyrins it 

 thus becomes very important to establish, if possible, the non-enzymic rate of reaction 

 between porphyrin and metal ion. Such studies might, for instance, elucidate the 

 mechanism of the enzymic insertion reaction and might also account for the metal 

 specificity, i.e. Fe++ vs Mg+''", exhibited by the cyclic tetrapyrroles found in living 

 tissues. 



The work of H. Fischer (Fischer and Orth, 1937) and others has established that 

 iron is more readily removed from (Morell and Stewart, 1956) and inserted into the 

 porphyrin ring when the metal ion is in the ferrous state. In hot glacial acetic acid 

 an equilibrium is apparently established between porphyrin and ferrous iron. Thus 

 if a porphyrin is heated in acetic acid and a reducing agent added then the metal is 

 removed; heating the free porphyrin with a great excess of ferrous acetate in acetic 

 acid leads, on the other hand, to a very rapid insertion of iron. The use of acetic acid 

 for the synthesis experiments is noteworthy since this organic acid is probably too 

 weak to charge the nitrogen atoms of the pyrrole nuclei. By the same token, a rela- 

 tively strong acid, even a mineral acid, is desirable for dissociation of the haem iron. 



The slow reaction of iron with porphyrins under approximately physiological 

 conditions may be attributed to a number of reasons, among which we may mention 

 the following: 



(a) Structure of the ligand 



The rigid planarity of the porphyrin macrocycle restricts the iron to an attack 

 which must be directed from either exactly above or exactly below the plane of the 

 ring. This fact must at least contribute to the slow reaction rate, especially since it is 

 known that ferrous ion is attached to the porphyrin by mainly 'ionic' bonds (Pauling 

 and Coryell, 1936). 



(b) Insolubility and aggregation of the reactants 



Most porphyrins are either insoluble, or are at least rather severely aggregated, in 

 aqueous solution at neutral pH. Furthermore, under these conditions the competition 

 from hydroxyl ions for either Fe+^ or Fe+++ is enormous and both oxidation states 

 of iron will be present as their insoluble hydroxides. 



The chemical incorporation of iron into porphyrins which have been treated with 

 sodium amalgam has been described in some detail by Orlando (1958). Briefly, his 

 results show that very good yields of iron complex can be obtained instantly in those 

 cases where the reducing agent is allowed to act for only short periods on the porphyrin 

 prior to the addition of the ferrous ion. This reaction has been interpreted in terms 

 of a distortion of the planar configuration of the porphyrin with concomitant exposure 

 of the pyrrole nuclei. The investigation did not reveal, however, why the yields for 

 coproporphyrin were superior to those for some other common porphyrin compounds. 



The combination of ferrous ion with porphyrins has been studied by Heikel, 



