Crosstalk between metal ions in Bacillus subtilis ferrochelatase.
Ferrochelatase (EC 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of Fe2+ into protoporphyrin IX, generating heme. In vitro assays have shown that all characterized ferrochelatases can also incorporate Zn2+ into protoporphyrin IX. Previously Zn2+ has been observed at an inner metal binding site close to the porphyrin binding site. Mg2+, which stimulates Zn2+ insertion by Bacillus subtilis ferrochelatase, has been observed at an outer metal binding site. Exchange of Glu272 to a serine eliminated the stimulative effect of Mg2+. We found that Zn2+ quenched the fluorescence of B. subtilis ferrochelatase and this quenching was used to estimate the metal affinity. Trp230 was identified as the intrinsic fluorophore responsible for the observed quenching pattern. The affinity for Zn2+ could be increased by incubating the ferrochelatase with the transition state analogue N-methyl mesoporphyrin IX, which reflected a close collaborative arrangement between the two substrates in the active site. We also showed that the affinity for Zn2+ was lowered in the presence of Mg2+ and that bound Zn2+ was released upon binding of Mg2+. In the ferrochelatase with a Glu272Ser modification, the interaction between Zn2+ and Mg2+ was abolished. It could thereby be demonstrated that the presence of a metal at one metal binding site affected the metal affinity of another, providing the enzyme with a site that regulates the enzymatic activity.
Hansson
Mattias D
MD
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, 221 00, Lund, Sweden. mattias.hansson@biokem.lu.se
Lindstam
Mats
M
Hansson
Mats
M
D001412:Bacillus subtilis / Q000201:enzymology*; D001483:Base Sequence; D002412:Cations; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005294:Ferrochelatase / Q000737:chemistry* / Q000235:genetics; D008670:Metals / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D002412:Cations; D017931:DNA Primers; D008670:Metals; D005294:Ferrochelatase