A novel thermostable hemoglobin from the actinobacterium Thermobifida fusca

Abstract

The gene coding for a hemoglobin‐like protein (Tf‐trHb) has been identified in the thermophilic actinobacterium Thermobifida fusca and cloned in Escherichia coli for overexpression. The crystal structure of the ferric, acetate‐bound derivative, was obtained at 2.48 Å resolution. The three‐dimensional structure of Tf‐trHb is similar to structures reported for the truncated hemoglobins from Mycobacterium tuberculosis and Bacillus subtilis in its central domain. The complete lack of diffraction patterns relative to the N‐ and C‐terminal segments indicates that these are unstructured polypeptides chains, consistent with their facile cleavage in solution. The absence of internal cavities and the presence of two water molecules between the bound acetate ion and the protein surface suggest that the mode of ligand entry is similar to that of typical hemoglobins. The protein is characterized by higher thermostability than the similar mesophilic truncated hemoglobin from B. subtilis, as demonstrated by far‐UV CD melting experiments on the cyano‐met derivatives. The ligand‐binding properties of Tf‐trHb, analyzed in stopped flow experiments, demonstrate that Tf‐trHb is capable of efficient O2 binding and release between 55 and 60 °C, the optimal growth temperature for Thermobifida fusca.

Protein structure in the truncated (2/2) hemoglobin family

“…(Ss-2/2HbN) (10 -12), four group II HbOs from M. tuberculosis (Mt-2/ 2HbO) (13), from Bacillus subtilis (Bs-2/2HbO) (14), from Thermobifida fusca (15), from Geobacillus stearothermophilus (16), and one group III HbP from C. jejuni (Cj-2/ 2HbP) (17).…”

Unusual proximal heme pocket geometry in the deoxygenated Thermobifida fusca: A combined spectroscopic investigation

“…In this framework, an investigation on the spectroscopic properties of the ferrous unliganded truncated hemoglobin from the actynomyces Thermobifida fusca (Tf-trHb) [16] has been undertaken with the aim of characterizing the fine structural properties of the Fe-histidine moiety as compared to ferrous unliganded horse myoglobin (h-Mb). The results of this study bring out relevant differences in the structure of the active site between Tf-trHb and h-Mb.…”

“…Previous investigations have shown that for a quantitative EXAFS analysis of hemeproteins MS fourbody terms have to be accounted for [13][14][15][16]20]. The inclusion of these higher order contributions is essential to obtain a good agreement between theoretical and experimental data.…”

“…However, inspection of the visible spectra suggests that the overall electronic structure of the porphyrin π system is not superimposable. In fact, in Tf-trHb, the peak position of the deoxygenated derivative is at 432 nm and hence red shifted with respect to h-Mb (435 nm) [16]. The Soret peak energies are affected by the overall electron density of the heme thus suggesting that there is a small but significant electron withdrawing effect (which enhances the energy of the π → π* transitions), in TftrHb with respect to h-Mb.…”

“…In the case of Tf-trHb, the observed blue shift of the Soret band, due to decreased π electron density, might be ascribed mainly to the short iron histidine bond but in the absence of concomitant hydrogen bonding donation to the N δ of the proximal ligand (see also next paragraph) and hence without the "electron push" effect. In turn, the effect of vinyl conjugation still needs to be investigated in detail by appropriate resonance Raman experiments, although no major "out of plane" shift of the double bonds is evident from inspection of the available X-ray structure [16]. In summary, the inverse relationship between the red shifted band III and blue shifted Soret spectrum in Tf-trHb might find a rationale in terms of electronic configuration only assuming an electron withdrawal effect by the proximal imidazole ring, fully protonated at the N δ .…”