INSTITUTO DE QUIMICA, FISICA DE LOS MATERIALES, MEDIOAMBIENTE Y ENERGIA
Unidad Ejecutora - UE
Heme pocket structural properties of a bacterial truncated hemoglobin from Thermobifida fusca
DROGHETTI, E.; NICOLETTI, F.P.; BONAMORE, A.; BOECHI, L.; ARROYO MAÑEZ, P.; ESTRIN, D.A.; BOFFI, A.; SMULEVICH, G.; FEIS, A.
AMER CHEMICAL SOC
Año: 2010 vol. 49 p. 10394 - 10394
ABSTRACT:An acidic surface variant (ASV) of the truncated hemoglobin from Thermobifida fusca wasdesigned with the aim of creating a versatile globin scaffold endowed with thermostability and a high level ofrecombinant expression in its soluble form while keeping the active site unmodified. This engineered proteinwas obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamicssimulations showed that the mutated residues remain solvent-exposed, not affecting the overall proteinstructure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in whichone, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] weresubstituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy andcompared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of themutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the COcomplexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm-1 and form2 with ν(FeC) and ν(CO) at 518 and 1920 cm-1, respectively. Molecular dynamics simulations were performedfor the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computationalresults demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 inform 2. TyrB10 does not directly interact with the bound CO.