title: Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme.
abstract: Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the haem biosynthetic pathway, catalyses the chelation of Fe(II) into the protoporphyrin IX ring. The energetics of the binding between murine ferrochelatase and mesoporphyrin were determined using isothermal titration calorimetry, which revealed a stoichiometry of one molecule of mesoporphyrin bound per protein monomer. The binding is strongly exothermic, with a large intrinsic enthalpy (DeltaH=-97.1 kJ x mol(-1)), and is associated with the uptake of two protons from the buffer. This proton transfer suggests that hydrogen bonding between ferrochelatase and mesoporphyrin is a key factor in the thermodynamics of the binding reaction. Differential scanning calorimetry thermograms indicated a co-operative two-state denaturation process with a single transition temperature of 56 degrees C for wild-type murine ferrochelatase. An increase in the thermal stability of ferrochelatase is dependent upon mesoporphyrin binding. Similarly, murine ferrochelatase variants, in which the active site Glu-289 was replaced by either glutamine or alanine and, when purified, contained specifically-bound protoporphyrin, exhibited enhanced protein stability when compared with wild-type ferrochelatase. However, in contrast with the wild-type enzyme, the thermal denaturation of ferrochelatase variants was best described as a non-co-operative denaturation process.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Franco
authors.forename: Guangyue
authors.initials: G
authors.lastname: Bai
authors.forename: Vesna
authors.initials: V
authors.lastname: Prosinecki
authors.forename: Filipa
authors.initials: F
authors.lastname: Abrunhosa
authors.forename: Gloria C
authors.initials: GC
authors.lastname: Ferreira
authors.forename: Margarida
authors.initials: M
authors.lastname: Bastos
mesh_terms: D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology; D001665:Binding Sites; D002021:Buffers; D002152:Calorimetry, Differential Scanning; D004795:Enzyme Stability; D005294:Ferrochelatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D008652:Mesoporphyrins / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D011522:Protons; D011524:Protoporphyrins / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics
chemical_list: D002021:Buffers; D008652:Mesoporphyrins; D011522:Protons; D011524:Protoporphyrins; D018698:Glutamic Acid; C031468:mesoporphyrin IX; C028025:protoporphyrin IX; D005294:Ferrochelatase