The Enzymic Incorporation of Iron into Protoporphyrin 215 



require two hydrogen atoms to be displaced for metal incorporation to occur and it 

 is likely that the reaction would be rapid. 



Wang : I would like to call attention to some work done by E. B. Fleischer at Yale which 

 may be related to these problems. As I have mentioned (p. 38), the rate of 'swallowing- 

 up' of the metal ions by porphyrin derivatives is strongly solvent-dependent. This is 

 not too surprising, since in 'swallowing' the metal ion, the porphyrin also peels off 

 most of the solvation of the ion. For example, we found that in acetone solution, 

 Cu++, Bi+++, Hg^"+ and Cd '^+ react readily with the dimethyl ester of protoporphyrin 

 to form the corresponding metallo-porphyrins, even at room temperature. But in 

 aqueous solutions there was no detectable reaction at the same temperature. Since 

 the biochemical incorporation of metal ions into porphyrins takes place in aqueous 

 solution, special provisions have to be made by the catalytic system to furnish faster 

 reaction paths. It appears possible that the main function of the enzyme systems is to 

 replace the attached water molecules and modify the ligand environment of the metal 

 ion in such a manner, though off-hand I do not know how, as to facilitate the 

 'swallowing-up' of the metal ion by the porphyrin. 



DwYER : The mechanism of incorporation of iron brings to mind some recent experiments 

 carried out at Canberra. The sexadentate molecules 1 : 2-propylenediaminetetraacetic 

 and cyclohexanediamine-tetraacetic acids in the optically active forms are completely 

 stereospecific in their metal complexes, i.e. the D configuration of the complex can 

 contain only the / configuration of the organic molecule, and vice versa. The metal 

 complex, e.g. Dl exchanges with inversion at a rate dependent on pH with the ligand 

 of the opposite configuration: Dl + d-* Ld + I. The reaction can be followed easily 

 in a polarimeter. The ferric complex exchanges its / ligand for the d ligand in less 

 than one minute at pH 6, and 25°C. In the reaction the / ligand gradually detaches 

 point by point and the d ligand enters by attachment to the vacated co-ordination sites. 



On the Enzymic Incorporation of Iron 



Falk : Perhaps attention should be drawn to the evidence (Dresel and Falk, Biochem. J. 

 63, 388, 1956), that protoporphyrin itself does not behave as though it is a true inter- 

 mediate in haem synthesis in chicken erythrocytes. We suggested in that paper that 

 some reduced derivatives of proto-, copro- and uro-porphyrins might be the true 

 intermediates. That this is true for uro- and copro-porphyrins has recently been 

 demonstrated directly by Granick and Mauzerall (/. biol. Chein. 232, 1141, 1958), 

 and there is as yet no evidence contrary to our suggestion that a reduced form of 

 protoporphyrin is the true intermediate. 



Margoliash: How do the results obtained by Neve compare with those of Schwartz, 

 Hill, Cartwright and Wintrobe {Fed. Proc. 18, 545, 1959)? These authors have shown 

 that both globin and protoporphyrin are required before any radioactive iron 

 is incorporated into haemoglobin. In their system, it does not seem that the globin 

 acts as an agent to keep either the porphyrin or the iron in solution, but rather that 

 the enzyme responsible for iron incorporation will function only on the preformed 

 globin-protoporphyrin complex. 



Neve: The ammonium sulphate purification shows an increase in specific activity, sug- 

 gesting that the protein is acting more as an enzyme than as a solubilizing agent to 

 the protoporphyrin and iron. 



The Tween detergent is not responsible for iron incorporation as demonstrated 

 in this paper. In addition, Phillips (this symposium, p. 32) while finding metal- 

 porphyrin complexing with a variety of Tweens and metals was unable to show any 

 chelation of iron with protoporphyrin. 



George: Haemoglobin derivatives are particularly insoluble in strong phosphate buffer 

 at a pH of about 6: perhaps this property could be utilized in the enzyme purification 

 to remove haemoglobin, as is done in the preparation of myoglobin. 



Falk: As I see it, the essential problem with systems incorporating Fe into haem is to 

 establish that one really has an enzymic system. There are certain incidental require- 

 ments: that the iron be maintained in solution, that sufficient of it be in the ferrous 



