Heme Pocket Structural Properties of a Bacterial Truncated Hemoglobin from Thermobifida fusca

ABSTRACT An acidic surface variant (ASV) of the “truncated” hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and high recombinant expression level in soluble form, while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe 107 and Arg 91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two or three of the conserved polar residues: TyrB10(54), TyrCD1(67), TrpG8(119), were substituted by Phe. Mutants were characterized by infrared and resonance Raman spectroscopy, and compared with the wild-type protein. Similar Fe-(proximal His) stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with (FeC)/(CO) at 509/1938 cm-1, and form 2 with (FeC)/(CO) at 518/1920 cm-1. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1, whereas it interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO.