ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P47. Diiron-containing Metalloprotein: Structural and Functional
Characterization of DF3, a Catalytic Model
M. Faiella a , C. Andreozzi a , O. Maglio a , V. Pavone a , W. F. DeGrado b , F. Nastri a ,
A. Lombardi a
a
Department of Chemistry, University of Napoli Federico II, Via Cinthia 80126 Napoli, Italy,
e-mail: angelina.lombardi@unina.it
b
Department of Biochemistry & Biophysics, University of Pennsylvania , 19104-6059 Philadelphia, PA, – USA
Diiron-oxo proteins are a class of macromolecules that catalyze different reactions, from ferroxidation to
hydroxylation, despite the same structural motif, the four-helix bundle [1].
To elucidate how the protein matrix tunes the properties of a single metal cofactor to obtain such a wide diversity
of functions, we designed minimal models called DFs [2]. The first model DF1 was de novo designed from a
retro-structural analysis of 6 different carboxylate-bridged diiron proteins. DF1 is made up of two 48-residue
helix-loop-helix (α2) motifs, able to specifically self-assemble into an antiparallel four-helix bundle, with a
Glu4His2 liganding environment for the diiron center, housed within the center of the structure. Although DF1
adopted the intended helical conformation and was able to coordinate different metal ions, it showed low water
solubility and did not support any catalytic activity. DF1 structural characterization [3] suggested that the interhelical
loop adopted a strained conformation, which may account for the molecule insolubility, while a pair of
symmetrically related hydrophobic Leu residues at position 13 of the sequence blocked the access to the active
site, preventing any activity.
Here we present DF3, a new DF model, designed to improve thermodynamic and functional properties of DF1.
The mutations in DF3 sequence are:
1. two glycine residues in position 9 and 13 to allow easy access of exogenous ligands to the metal site (Figure);
2. a new inter-helical loop, introduced to evaluate its influence on the overall folding and solubility.
DF3 is highly water soluble and the NMR characterization of the Zn(II)-complex [4] reveals that it is able to fold
into a stable native-like four-helix bundle structure. The loop region is very-well defined and adopts a unique
conformation. DF3 binds Co, Mn and Zn in the expected stoichiometry and coordination geometry. Chemical
denaturation, by Gdn·HCl, shows that Leu 9 and 13 substitutions destabilize the protein, as expected;
nevertheless, the newly designed loop positively affects the thermodynamic properties of DF3.
UV-Vis characterization reveals that DF3 reversibly oxidizes Fe(II) to Fe(III) and its diferric complex is stable in
water at pH=7. Kinetic experiments were performed to explore the catalytic activity of di-Fe(III)-DF3 using
different substrates, demonstrating a high selectivity in diphenolase reactions.
These data confirm that DF3 is a promising candidate in the development of catalytic artificial proteins.
Active site cavity in DF3
References:
[1] S.J. Lange and L. Que, Jr., Curr. Opin. Chem. Biol., 2, 159 (1998).
[2] O.Maglio, F. Nastri, R.T.M. de Rosales, M. Faiella, V. Pavone, W.F. DeGrado, A. Lombardi, Comptes
Rendus – Chimie, 10, 703 (2007).
[3] A. Lombardi, C.M. Summa, S. Geremia, L. Randaccio, V. Pavone, W.F. DeGrado, Proc. Natl. Acad. Sci.
USA, 97, 6298 (2000).
[4] Faiella et al. manuscript in preparation
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