Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme

• Type: Article in International Scientific Journal
• Date: 2005

Authors

Franco, R (Author) Other; Bai, GY (Author) Other; Prosinecki, V (Author) Other; Abrunhosa, F (Author) Other; Ferreira, GC (Author) Other; Bastos, M (Author) FCUP

Journal

Title: Biochemical Journal

Vol. 386

Pages: 599-605

ISSN: 0264-6021

Publisher: PORTLAND PRESS LTD

Indexing

ISI Web of Knowledge - 11 Citations

Scopus - 11 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

Other information

Authenticus ID: P-000-47S

DOI: 10.1042/bj20040921

Abstract (EN): 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 (Delta H = -97.1 kJ(.)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 wildtype enzyme, the thermal denaturation of ferrochelatase variants was best described as a non-co-operative denaturation process.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 7