The Enzymic Incorporation of Iron into Protoporphyrin 213 



light absorption at 550 m/< ; the NagSaOi appeared to be responsible for this effect. 

 Thus although it is difficult to calculate the precise amount of haem synthesized in 

 these experiments, the rate appears to be somewhat less than 2 //moles/I. hr. 



The presence of Na2S204 was required in order to prevent the development of an 

 incipient turbidity. Experiments carried out under Nj or in which the dithionite 

 was added after a period of about one hour showed that approximately the same 

 amount of haem had been formed in the absence of the reducing agent. It thus 

 appears that NagSjOj is neither inhibitory (apart from bleaching of the porphyrin 

 solution) nor is it essential for the reaction. 



The use of higher concentrations of pyridine (supplied as the sulphate) or the 

 substitution of imidazole for pyridine did not increase the rate. 



Although porphyrin c in neutral aqueous solution exhibits a four-banded spectrum, 

 the bands are not clearly separated and considerable aggregation is indicated. Con- 

 sequently, sodium lauryl alcohol sulphonate was added as a dispersing agent. Although 

 this reagent greatly strengthens and sharpens the visible absorption bands of porphyrin 

 c, the iron-incorporation rate was diminished, possibly as a consequence of com- 

 plexing of the iron by the added detergent. Some further experiments were carried 

 out with cationic detergents but these likewise did not augment the reaction rate. 



In summary, therefore, the reaction rate observed in the system given above is the 

 maximum that it has been possible to obtain up to the present time. 



Studies were conducted on the rate of uptake of several divalent ions (other than 

 ferrous) by a 1-7 x 10~* m porphyrin c solution in 0-1 m acetate buffer pH 5-7. The 

 cupric ion gave a very insoluble precipitate which dissolved with difficulty in butanol 

 and exhibited a turacin-like spectrum. The zinc ion, as judged by the rapid loss of the 

 porphyrin band at 510 m/z, also reacted at a rapid rate. The two-banded spectrum 

 of the zinc complex resembled that of the cupric complex except that in the former 

 case the less intense band lay at the longer wavelength. 



Incorporation of Divalent Ions into Porphyrin Cytochrome c 



Fe++, Mn++ and Co++ were found to react very slowly with porphyrin cytochrome c. 

 The cupric ion reacted rapidly and the zinc ion entered the porphyrin-protein at a 

 moderate rate. The presence of the latter two metal ions inside the porphyrin ring 

 was confirmed by spectral examination after dialysis of the new metallo-porphyrin 

 proteins against glycine solution. 



DISCUSSION AND CONCLUSIONS 



Even under the most favourable conditions found, the rate of non-enzymic haem 

 synthesis is only approximately equal to the average rate of synthesis of cytochrome c 

 haem/l.hr. by a culture of Ustilago sphaerogena. In the case of the microbial reaction, 

 the ligand concentration at the beginning of a 10 hr fermentation period is essentially 

 zero. It thus appears highly improbable that any significant quantity of haem will be 

 formed within living cells by a purely non-enzymic reaction. 



The fact that both cupric and zinc ions react rapidly with porphyrin c suggests that 

 aggregation in solution is not the most important factor limiting the rate for ferrous 

 iron. The generally slower reaction of the divalent metal ions with porphyrin cyto- 

 chrome c may be attributed to the known inaccessibility of the prosthetic group in 

 this enzyme. 



It is tempting to speculate on the mechanism of the relatively rapid reaction involved 

 in those cases in which either partially-reduced porphyrins (so-called semi-porphyri- 

 nogens; Orlando, 1958) or higher temperatures have been employed for the synthesis. 

 In particular, it would be interesting to know if these treatments (sodium amalgam; 

 heat) lead to the temporary establishment of a more nearly octahedral orientation of 

 the ligand atoms. If such a mechanism were indeed responsible in those few cases in 

 which the chemical reaction will proceed at room temperature then it might be 

 possible, with some adaptation, to apply the same mechanism to the enzymic synthesis. 



