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 (ΔH=−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 °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.
Skip Nav Destination
Article navigation
March 2005
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
March 08 2005
Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme
Ricardo FRANCO;
Ricardo FRANCO
*Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal
Search for other works by this author on:
Guangyue BAI;
Guangyue BAI
†CIQ(UP), Departamento de Química, Faculdade de Ciências da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal
Search for other works by this author on:
Vesna PROSINECKI;
Vesna PROSINECKI
*Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal
Search for other works by this author on:
Filipa ABRUNHOSA;
Filipa ABRUNHOSA
†CIQ(UP), Departamento de Química, Faculdade de Ciências da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal
Search for other works by this author on:
Gloria C. FERREIRA;
Gloria C. FERREIRA
1
‡Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, U.S.A.
§H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL 33612, U.S.A.
Search for other works by this author on:
Margarida BASTOS
Margarida BASTOS
1
†CIQ(UP), Departamento de Química, Faculdade de Ciências da Universidade do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
June 02 2004
Revision Received:
October 07 2004
Accepted:
October 21 2004
Accepted Manuscript online:
October 21 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2005
Biochem J (2005) 386 (3): 599–605.
Article history
Received:
June 02 2004
Revision Received:
October 07 2004
Accepted:
October 21 2004
Accepted Manuscript online:
October 21 2004
Citation
Ricardo FRANCO, Guangyue BAI, Vesna PROSINECKI, Filipa ABRUNHOSA, Gloria C. FERREIRA, Margarida BASTOS; Porphyrin-substrate binding to murine ferrochelatase: effect on the thermal stability of the enzyme. Biochem J 15 March 2005; 386 (3): 599–605. doi: https://doi.org/10.1042/BJ20040921
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.