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Ferrochelatase of Rhodopseudomonas spheroides.

ABSTRACT: Extracts of Rhodopseudomonas spheroides contain two ferrochelatases: one is soluble and forms metalloporphyrins from deuteroporphyrin and haematoporphyrin; the other is particulate and forms metalloporphyrins from protoporphyrin, mesoporphyrin, deuteroporphyrin and haematoporphyrin. Neither enzyme incorporates Mg(2+) into porphyrins or Fe(2+) into porphyrin cytochrome c. By using the particulate enzyme, plots of 1/v versus 1/s when one substrate was varied and the other kept constant showed that neither substrate affected the K(m) of the other. The suggested sequential mechanism for the reaction is supported by derivative plots of slopes and intercepts. The K(m) for deuteroporphyrin was 21.3mum and that for Co(2+) was 6.13mum. The enzyme incorporated Co(2+), Fe(2+), Zn(2+), Ni(2+) and Mn(2+); Cd(2+) was not incorporated and was an inhibitor, competitive with respect to Co(2+), non-competitive with respect to deuteroporphyrin. The K(i) for Cd(2+) was 0.73mum. Ferrochelatase was inhibited by protohaem, non-competitively with respect to Co(2+) or with respect to deuteroporphyrin. Inhibition by magnesium protoporphyrin was non-competitive with respect to deuteroporphyrin, uncompetitive with respect to Co(2+). The inhibitory concentrations of the metalloporphyrins are lower than those required for the inhibition of delta-aminolaevulate synthetase by protohaem. Fe(2+) is not incorporated aerobically into porphyrins unless an electron donor, succinate or NADH, is supplied; the low aerobic rate of metalloporphyrin synthesis obtained is insensitive to rotenone and antimycin. The rate of Fe(3+) incorporation increases as anaerobic conditions are achieved.

SUBMITTER: Jones MS

PROVIDER: S-EPMC1179374 | biostudies-other | 1970 Sep

REPOSITORIES: biostudies-other

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Localization of ferrochelatase and of newly synthesized haem in membrane fractions from Rhodopseudomonas spheroides.

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Project description:1. Uroporphyrinogen I synthetase of Rhodopseudomonas spheroides was purified more than 200-fold from the soluble protein of broken bacterial cells. The enzyme had molecular weight 36000, an isoelectric point of 4.46 and migrated as a single active protein band on disc-gel electrophoresis at pH7.5 and 8.9. 2. The enzyme consumed porphobilinogen and formed uroporphyrinogen at pH8.2 without the accumulation of intermediates. In the presence of hydroxylamine, ammonia or methoxyamine the production of porphyrinogen was inhibited and the enzyme formed open-chain polypyrroles instead. 3. These polypyrroles behaved like uroporphyrinogen on Sephadex G-25; they were colourless and had unsubstituted alpha-pyrrolic positions. The inhibitory amines were incorporated into the molecules. 4. The polypyrroles formed porphyrins non-enzymically and the cyclization reaction was accompanied by the release of the inhibitory amine. Exchange of the amino function of the original porphobilinogen in the polypyrrole was complete with hydroxylamine and almost complete with methoxyamine, both ammonia and methoxyamine being present in the polypyrrolic material. 5. The behaviour, properties and composition of the radioactive hydroxylamine derivative were consistent with a tetrapyrrolic structure, probably a pyrrylmethane, that was not cyclized, rather than with di-, tri- or penta-pyrrolic structures. No monopyrrolic or dipyrrolic Ehrlich-positive material was released on cyclization. The ammonia and methoxyamine derivatives had properties similar to the hydroxylamine derivative. 6. Another modified pyrrole was detected only in experiments with hydroxylamine. It differed from both porphobilinogen and known dipyrroles and appeared to be a monopyrrole. 7. The participation of positively charged reaction centres in the enzymic mechanism, particularly in the cyclization step, is discussed.

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16s and 23s components in the ribosomal ribonucleic acid of Rhodopseudomonas spheroides.

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