Heme-linked groups

Chance found that pH has little effect on the dissociation constants in the range 3.6 to 8.8, which suggests that neither of the two heme-linked groups found by Theorell and Paul (15) affects the ability of peroxidase to form its enzyme substrate complexes. On the basis of this observation he suggests that the primary combination takes the form ... 

Theorell, H. 1947. Heme-linked groups and mode of action of some hemoproteins. Advances in Emymol. 7, 265. 

Theorell, H., Heme-Linked Groups and Mode of Action of Some Hemoproteins, in F. F. Nord, ed., Advaruxs in Enzymology, Vol. VII, Interscience, New York-London, 1947, p. 266. 

Theorell, Hugo, Heme-Linked Groups and Mode of Action of Some... 

Hemoproteins, Mode of Action, and Heme-Linked Groups (Theorell). VII 265... 

HEME-LINKED GROUPS AND MODE OF ACTION OF SOME HEMOPROTEINS... 

In accordance with this theory, one has to seek the cause of the different modes of reaction of the hemoproteins mainly in their heme-linked groups. A number of methods may be resorted to for the determination of the nature of these groups ... 

It is only through the explanation of the chemical nature of heme-linked groups in the hemoglobin that it has become possible to understand this effect. 

Determinations of the dependence of the oxidation-reduction potential upon the pH of the solution are in many cases able to give data concerning the dissociation constants for heme-linked groups according to the general equation ... 

Coryell and Pauling (22) have attempted a structural interpretation of the heme-linked groups in hemoglobin and its derivatives on the basis of the evidence mentioned above. They summarize the heme-linked groups in the pH range 4.5 to 9 as follows ... 

Ferroq/tochrome c shows the same bands in the spectroscope over the entire pH scale. Precise spectrophotometric measurements of the light absorption at different acidities seem not to have been carried out. Unpublished experiments by K. G. Paul show that the absorption coefficient at 550 mp, on top of the main visible band, changes its value somewhat between pH 9 and 10, indicating a heme-linked group with pK in this region. 

There are, however, also other reasons in support of the assumption that heme-linked groups in ferrocytochrome are affected by changes in pH. Below pH 4 and above pH 13 ferrocytochrome c is autoxidizable. Between pH 3 and pH 12 no carbon monoxide compounds are formed but at pH 2... 

In order to study the dissociation constants of heme-linked groups, it was obviously of great interest to determine the redox potential throughout the whole pH scale. In the author s institute, Paul has carried out experiments along these lines (52). Owing to experimental difficulties, the pH... 

This result indicates the existence of a hitherto unknown heme-linked group in ferricytochrome, with pK 0.86 (see Fig. 3). 

This assumption explained also another peculiar fact, i.e., how heme-linked groups could be titrated without breaking their bonds to the iron. 

It may further be pointed out that there is a contradiction between Russell and Pauling (1939) (56) and Coryell and Pauling (1940) (22). The former attributed the heme-linked group, pK 9.5, in imidazole hemiglobin to the titration of the imino group (see formula A). Coryell and Pauling,... 

As can be seen from the Table I (page 266), four peroxidases have been produced in a pure or nearly pure state. Two of them have been crystallized, horse-radish peroxidase and lactoperoxidase these are the only ones that have been submitted to any investigations concerning their heme-linked groups. Most of the work has been carried out with horse-radish peroxidase, which is comparatively easily available and has the great advantage that it can be split reversibly into prosthetic group and protein component (71). 

Careful spectrophotometric measurements on horse-radish peroxidase at 655 mp and different pH values revealed the existence of a heme-linked group with pK of 4.0. Its dissociation curve coincides with the activity measurements (purpurogallin tests) from pH 7 to pH 3, see Figure 7. 

Differential titration experiments were carried out on three systems (79). Ferroperoxidase and carbon monoxide ferroperoxidase gave identical curves. The peroxidase thus shows no Bohr effect. Since the two derivatives correspond as to bond types to the analogous hemoglobin and carbon monoxide hemoglobin, it can be stated with fair certainty that the heme-linked groups in horse-radish peroxidase are not imidazole. 

On the other hand, we lack an explanation for the heme-linked group with pK 7 in ferroperoxidase. It cannot be a hydroxyl bound to the ferrous iron, because such a hydroxyl should be displaced by carbon monoxide and appear in the titration of ferroperoxidase against carbon monoxide ferroperoxidase. This was not the case, and furthermore it seems hard to imagine how a hydroxyl group could be bound to ferroheme by ionic bonds. 

Attempts to determine the nature of the heme-linked groups in catalases were met with greater difficulties than in any other kind of hemo-proteins. This is due to several unfavorable circumstances catalase hematin cannot be reduced by ordinary reducing agents catalases cannot be split reversibly the interpretation of many different data is obscured by interaction phenomena between the four iron porphyrins in each molecule. Furthermore, until recently only liver catalases with two different kinds of colored groups were available. This last drawback is now overcome with the isolation of easily available catalases containing only hematin and protein. 

For the reasons mentioned above, differential titration and redox potential determinations cannot be used to determine the nature of the heme-linked groups. Under such conditions it seems impossible to solve the whole question of the heme-linked groups of catalases by the aid of the methods we have at our immediate disposal. Some amino acid analyses have been carried out, but these cannot yet contribute to our knowledge of the heme-linked groups. However, some information has been obtained from magnetometric observations, from spectrophotometric studies of different catalase compounds with inhibitors, and from activity determinations in solutions of different pH values and varying concentrations of inhibiting anions. 

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