Biosynthesis of hematin compounds in a hemin requiring strain of Micrococcus pyogenes var. aureus. I. The significance of coenzyme A for the terminal synthesis of catalase.

A mutant of Micrococcus pyogenes var. aureus isolated in 1952 has been used to investigate the biosynthesis of certain hematin enzymes. This strain has been described and its hemin requirements analysed in a series of papers (Jensen and Thofern 1953a, b, c, d; 1954). Since the present study is very closely related to and based upon the previous results, a brief summary is indicated: (1) The hemin requirement is not absolute, since in brain-heart infusion broth and similar liquid media hemin is not required for growth. On the surface of solid media, however, or in aerated liquid media the strain grows only in the presence of hemin (ferriprotoporphyrin chloride). (2) If grown in the absence of hemin in broth the bacteria are completely devoid of their hematin compounds (catalase, cytochromes, cytochrome oxydase). These compounds are always formed by the parent strain but are synthesized by the hemin-requiring mutant only if hemin or a suitable hemin source is present in the culture medium. The absence of the hematin compounds is reflected by the lack of catalase activity, the lack of cytochrome respiration, and the absence of the typical absorption bands associated with the cytochromes. (3) If resting-cell suspensions of bacteria grown without hemin are supplied with hemin the hematin compounds are formed. This formation occurs within 15 to 20 min (at 37 C) after the addition of hemin. The resulting enzyme activities are of the same magnitude as those of the parent strain or those of the mutant grown in the presence of hemin. Growing in the absence of hemin the cells of the hemin-requiring mutant synthesize the apoenzymes or protein moieties of their hematin compounds. Supplied with hemin they then perform the terminal synthesis which by linking the prosthetic group with the apoenzymes produces the active holoenzymes. (4) The terminal synthesis of catalase and other hematin compounds is not significantly influenced by electrostatic forces. (5) Protoporphyrin cannot take the place of hemin but mesoor deutero-hemin can. (6) Certain metal-porphyrins inhibit the terminal synthesis of the cytochrome system but not the terminal synthesis of catalase. Some hematin compounds such as horseradish peroxydase or hemoglobin can be split reversibly, that is, their prosthetic group (hemin) can be separated from the protein which then can be isolated and purified without losing its ability to reunite with hemin in vitro. Since this can be done with highly purified hematin compounds, the reunion seems to be a spontaneous reaction involving the two partners only and not requiring a "combining system." Catalase cannot be split reversibly. Though Agner (1935) claimed some activity on the addition of hemin to the dialyzed protein moiety, Tauber and Kleiner (1935) and Sumner and Dounce (1939) who repeated his experiments did not obtain any activity. Thus "the problem of splitting catalase reversibly into protein and hematin is still unsolved." (Theorell, 1951). Two interpretations regarding this failure are possible: (a) The hemin being firmly attached to the apocatalase cannot be split off without causing denaturation of the protein. (b) The recombination does not occur spontaneously but requires a combining enzyme or enzyme system. If a combining enzyme is required, reversible cleavage of crystalline catalase obviously would be impossible since highly purified catalase cannot contain such an enzyme. There is good reason to assume that the terminal synthesis of catalase in this particular case is enzymatically catalyzed.