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Aula de Seminarios INQUIMAE - DQIAQF
Facultad de Ciencias Exactas y Naturales
Ciudad Universitaria - Pab. 2  -  Piso 3

H-bonding interactions in the active site of heme proteins: the case of
Thermobifida fusca haemoglobin
G. Smulevich
Dipartimento di  Chimica “Ugo Schiff�, Università  di Firenze, Via della
Lastruccia 3,
 Firenze, I-50019, Italy
E.mail: giulietta.smulevich en unifi.it

Truncated hemoglobins (Hbs) belong to a family of small heme proteins that
is widely distributed among bacteria, protozoa and plants [1]. Their
sequences are 20–40 residues shorter than those of animal Hbs. They are
characterized by an unusual structural fold and a remarkable variability in
the nature of the distal heme pocket residues. These proteins can bind
oxygen with high affinity; however, their function is not yet fully
understood. Truncated Hbs are divided into three groups (I–III) that share
less than 30% sequence similarity [1].The  structural and functional
properties of the active site of the bacterial hemoglobin from *Thermobifida
fusca *(Tf-TrHb, Group II)* *are largely determined by three polar amino
acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a
combinatorial set of mutants, in each of which these three amino acids have
been singly, doubly, or triply replaced by a Phe residue, to perform a
detailed
study on H-bonding interactions between the protein and heme-bound ligand. A
combination of UV-Vis, and resonance Raman spectroscopies has been
particularly useful in highlighting the structural properties of these
proteins.  In the present work, characterization of H2S [2], CO [3]
and F-binding [4,5] to Tf-TrHb and to thecomplete group of mutants
where the three key amino acids TrpG8, TyrCD1,
and TyrB10, are progressively substituted with the non-hydrogen bonding
phenylalanine, will be presented.
A detailed picture of H-bonding in the distal cavity environment has been
obtained. It appears that while TrpG8 and TyrCD1 can form strong H-bonds
with the ligands, TyrB10 can only interact weakly.
REFERENCES
1.  D.A. Vuletich and J.T.J. Lecomte, *J. Mol. **Evol.* 62, 196–210 (2006).
2.  F.P. Nicoletti, A. Comandini, A. Bonamore, L. Boechi, F. Boubeta, A.
Feis, G. Smulevich, A. Boffi, *Biochemistry *(2010), 49, 2269–2278
3. E. Droghetti, F.P. Nicoletti, A. Bonamore, L. Boechi, P. Arroyo Manez,
D. A. Estrin, A. Boffi, G. Smulevich,‡and A. Feis, *Biochemistry *(2010)
, 49,
10394–10402
4. E. Droghetti, F. P. Nicoletti, A. Bonamore, N. Sciamanna, A. Boffi, A.
Feis, G. Smulevich,
*    J. of Inorg. Biochemistry* 105 (2011) 1338–1343
5. F.P. Nicoletti, E. Droghetti, L. Boechi, A. Bonamore, N. Sciamanna, D.
A. Estrin, A. Feis, A.   Boffi, and G. Smulevich,  *JACS*  (2011) under
revision