RESUMO
Protoporphyrin ferrochelatase catalyzes the insertion of Fe2+ into protoporphyrin IX to form heme. To determine whether a conserved, active site π-helix contributes to the translocation of the metal ion substrate to the ferrochelatase-bound porphyrin substrate, the invariant π-helix glutamates were replaced with amino acids with non-negatively charged side chains, and the kinetic mechanisms of the generated variants were examined. Analysis of yeast wild-type ferrochelatase-, E314Q- and E318Q-catalyzed reactions, under multi- and single-turnover conditions, demonstrated that the mutations of the π-helix glutamates hindered both protoporphyrin metalation and release of the metalated porphyrin, by slowing each step by approximately 30-50%. Protoporphyrin metalation occurred with an apparent pKa of 7.3⯱â¯0.1, which was assigned to binding of Fe2+ by deprotonated Glu-314 and Glu-314-assisted Fe2+ insertion into the porphyrin ring. We propose that unwinding of the π-helix concomitant with the adoption of a protein open conformation positions the deprotonated Glu-314 to bind Fe2+ from the surface of the enzyme. Transition to the closed conformation, with π-helix winding, brings Glu-314-bound Fe2+ to the active site for incorporation into protoporphyrin.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Ferroquelatase/química , Ferro/química , Protoporfirinas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Animais , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Ferroquelatase/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Humanos , Camundongos , Mutação , Estrutura Secundária de Proteína , Protoporfirinas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Frataxin is a mitochondrial iron-binding protein involved in iron storage, detoxification, and delivery for iron sulfur-cluster assembly and heme biosynthesis. The ability of frataxin from different organisms to populate multiple oligomeric states in the presence of metal ions, e.g. Fe(2+) and Co(2+), led to the suggestion that different oligomers contribute to the functions of frataxin. Here we report on the complex between yeast frataxin and ferrochelatase, the terminal enzyme of heme biosynthesis. Protein-protein docking and cross-linking in combination with mass spectroscopic analysis and single-particle reconstruction from negatively stained electron microscopic images were used to verify the Yfh1-ferrochelatase interactions. The model of the complex indicates that at the 2:1 Fe(2+)-to-protein ratio, when Yfh1 populates a trimeric state, there are two interaction interfaces between frataxin and the ferrochelatase dimer. Each interaction site involves one ferrochelatase monomer and one frataxin trimer, with conserved polar and charged amino acids of the two proteins positioned at hydrogen-bonding distances from each other. One of the subunits of the Yfh1 trimer interacts extensively with one subunit of the ferrochelatase dimer, contributing to the stability of the complex, whereas another trimer subunit is positioned for Fe(2+) delivery. Single-turnover stopped-flow kinetics experiments demonstrate that increased rates of heme production result from monomers, dimers, and trimers, indicating that these forms are most efficient in delivering Fe(2+) to ferrochelatase and sustaining porphyrin metalation. Furthermore, they support the proposal that frataxin-mediated delivery of this potentially toxic substrate overcomes formation of reactive oxygen species.
