Tài liệu Báo cáo khoa học: Binding of ligands originates small perturbations on the microscopic

Binding of ligands originates small perturbations on the

microscopic thermodynamic properties of a multicentre

redox protein

Carlos A. Salgueiro1,2, Leonor Morgado1,2, Bruno Fonseca1,2, Pedro Lamosa1

, Teresa Catarino1,2,

David L. Turner3 and Ricardo O. Louro1

1 Instituto de Tecnologia Quimica e Biolo´gica, Universidade Nova de Lisboa, Portugal

2 Departamento de Quimica da Faculdade de Cieˆncias e Tecnologia da Universidade Nova de Lisboa, Portugal

3 School of Chemistry, University of Southampton, UK

The structural aspects of protein complexes have

received considerable attention and several experimen￾tal and computational methods for the structural

determination of complexes exist [1]. Redox proteins

usually form transient complexes that can be studied

using NMR methods, which, in addition to the struc￾tural characterization, also provide information on the

lifetime and dynamics of the bound forms [2,3]. Trans￾fer of electrons between redox proteins at rates com￾patible with metabolic processes requires the proper

orientation of the partners for close approximation of

the redox centres of the donor and acceptor, and that

the reduction potentials ensure a favourable driving

force, which is one of the main determinants of the

rate of electron transfer [4]. Experimental measure￾ments of the reduction potentials of proteins involved

in complexes have been reported [5–7], but the effect

of partner binding on the microscopic properties of the

redox centres in proteins with multiple centres has not

been addressed in detail yet.

Cytochromes c3 from sulfate-reducing bacteria are

small soluble proteins containing four haems, and have

been assigned a fundamental role in the bioenergetic

metabolism of these organisms, mediating the flow of

electrons from periplasmic hydrogenases to respiratory

transmembrane electron transfer complexes coupled to

the transfer of protons [8–11]. Several cytochromes c3

have been isolated and characterized in great detail

with respect to structure (for a recent revision of struc￾tural work see [12]), equilibrium thermodynamic prop￾erties [9,13–17] and transient kinetic properties [17–19].

These studies have shown that cytochromes c3 have

the required thermodynamic properties to perform a

coordinated transfer of two electrons coupled to the

transfer of protons in agreement with their proposed

physiological role as partners of hydrogenase [8,20,21].

Keywords

cytochrome c3; electron transfer; NMR;

protein docking; thermodynamic properties

Correspondence

R. O. Louro, Instituto de Tecnologia

Quimica e Biolo´gica, Universidade Nova de

Lisboa, Rua da Quinta Grande 6,

2780-156 Oeiras, Portugal

Fax: 351-21-4428766

Tel: 351-21-4469848

E-mail: [email protected]

(Received 15 December 2004, revised 15

February 2005, accepted 7 March 2005)

doi:10.1111/j.1742-4658.2005.04649.x

NMR and visible spectroscopy coupled to redox measurements were used

to determine the equilibrium thermodynamic properties of the four haems

in cytochrome c3 under conditions in which the protein was bound to lig￾ands, the small anion phosphate and the protein rubredoxin with the iron

in the active site replaced by zinc. Comparison of these results with data

for the isolated cytochrome shows that binding of ligands causes only small

changes in the reduction potentials of the haems and their pairwise inter￾actions, and also that the redox-sensitive acid–base centre responsible for

the redox–Bohr effect is essentially unaffected. Although neither of the lig￾ands tested is a physiological partner of cytochrome c3, the small changes

observed for the thermodynamic properties of cytochrome c3 bound to

these ligands vs. the unbound state, indicate that the thermodynamic prop￾erties measured for the isolated protein are relevant for a physiological

interpretation of the role of this cytochrome in the bioenergetic metabolism

of Desulfovibrio.

Abbreviations

DvHc3, Desulfovibrio vulgaris (Hildenborough) cytochrome c3; DvHc3:Pi, Desulfovibrio vulgaris cytochrome c3 with phosphate; DvHc3:ZnRb,

Desulfovibrio vulgaris cytochrome c3 with zinc rubredoxin; EXSY, exchange spectroscopy.

FEBS Journal 272 (2005) 2251–2260 ª 2005 FEBS 2251