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Rufus Lumry 



imidazole nitrogens. The latter are said to occupy the fifth and sixth ligand 

 positions. Cytochrome c may be partially opened to replace the sixth -position 

 ligand, presumably an imidazole group, by a cyanide ion. The charge situ- 

 ation is then reversed as compared with the intact protein since the resulting 

 mixed iron complex of protein, porphyrin and cyanide ion has a negative 

 charge in the reduced state and no net charge in the oxidized state. If the 

 electrostatic effect is predominate, the potential should be shifted well over 

 in favor of the oxidized state perhaps to -250 mv, yet it shifts only from 

 + 260 mv. to -62 mv. Hence the electrostatic effect cannot be the total ex- 

 planation of the normal oxidation-reduction potential in this case and we must 

 look to the fifth -position ligand or the porphyrin of the mixed complex for a 

 more complete explanation. We are thus directed to the third possible explan- 

 ation for the behavior of protein reactions. 



The geometry of an isolated hemochrome or hemichronne complex is deter- 

 mined by the directed valences of the iron 3d orbitals. On incorporation of 

 these complexes into the protein matrix other factors will influence the geom- 

 etry of the complex. The local anisotropic electrostatic field can distort the 

 ligands from the "zero-order" geometry of the isolated complex though it is 

 hard to believe that the effect can be large. Of more importance, the thermo- 

 dynamic factors of protein stability may be best satisfied, i.e., the lowest 

 free energy of the total protein achieved, by distortion of the ligands from the 

 zero-order geometry. The porphyrin may be bent or twisted in hydrophobic 

 bonding and the fifth-position ligand and, in the case of cytochrome c, the 

 sixth-position ligand, which is also coupled to the protein chain, can be re- 

 oriented as a consequence of local folding details to produce bending, twisting, 

 compression or stretching of the nitrogen-iron bond. ^^' It is beginning to 

 look as though distortion of the porphyrin may not be too important in ground- 

 state electronic processes of the heme proteins. In preliminary Faraday- 

 effect experiments, Rosenberg* ' has found that there is no Faraday effect 

 associated with the absorption bands of cytochrome c except with the very 

 small band at 675 m|ji and the delta band at about 315 mfj.. According to 

 present understanding of the Faraday effect these results mean that electronic 

 states responsible for the pronninent Soret and visible bands do not contain 

 admixtures of 3d iron orbitals. There would thus appear to be no direct 

 coupling between these ir-system orbitals responsible for visible and Soret 

 absorption and the iron 3d orbitals, though of course there is cT-bonding to the 

 porphyrin nitrogen atoms. As Gouterman* ^ ■^' has pointed out, the effect of 

 metal ion on porphyrin spectra can be well explained by mixing of the iron p^ 

 orbital with the tt system. The chemical reactions, including the oxidation- 

 reduction process, involve essentially pure 3d orbital. These observations 

 suggest that the physiological reactions of iron are not strongly related to the 

 porphyrin it system and are not much influenced by p -position substitution of 

 the porphyrin or distortion of the porphyrin. It is not improbable that we shall 

 come to understand the rather large changes in the position and intensity of 

 the visible bands of porphyrin proteins during reaction to be the result in part 

 of direct distortions of the it system of the porphyrin brought about by the 

 forces due to the folding of the protein. Fleischer and colleagues* ^' have 

 recently demonstrated that the porphyrin plane can take on a number of confor- 

 mations in different metal-porphyrin densities. These probably result from 

 weak crystalline forces and lead us to believe that the porphyrin ring and thus 

 the IT system may be forced to take on many different conformations in differ- 

 ent proteins. In general it can be anticipated that these distortions will have 

 more influence on spectra than on the physiologically important electronic 

 properties of the bound nnetal ion at least for heme proteins. However, the 3d 

 orbitals of magnesium lie too high to be nnuch involved in the reactions of 



