Structural and functional investigation on 2/2HbO from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

GIORDANO, DANIELA; PESCE, ALESSANDRA; BUSTAMANTE, JUAN PABLO; HOWES, BARRY D.; BOECHI, LEONARDO; DI PRISCO, GUIDO; ESTRIN, DARÍO; NARDINI, MARCO; SMULEVICH, GIULIETTA; VERDE, CINZIA; BOLOGNESI, MARTINO

Congreso; 18th International Conference on Oxygen-Binding and Sensing Proteins; 2014

The genome of Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct truncated hemoglobins exhibiting the 2/2 alfa-helical fold and one flavohemoglobin. The unusually high number of Hbs strongly suggests that these proteins are bound to fulfil important physiological roles that are perhaps related to the peculiar features of an O2-rich and cold habitat. One of these globins, belonging to group II (Ph-2/2HbO), encoded by the PSHAa0030 gene, has been extensively characterized by spectroscopic analysis, kinetic measurements, computer simulation and very recently by X-ray diffraction. Recent results on a genomic mutant strain highlight the involvement of Ph-2/2HbO in the protection against the stress induced by high O2 concentration, hydrogen peroxide and nitrosating agents. Moreover, the protein was shown to catalyse peroxynitrite isomerisation in vitro. The heme cavity of Ph-2/2HbO is characterised by the presence of a Trp residue on the bottom of the heme distal pocket in position G8 and Tyr and His residues in position B10 and CD1, respectively. Electronic absorption and resonance Raman spectroscopies at pH 7.6 indicate that the protein displays hexa-coordination, both in the ferric and ferrous states. The 3D structure of ferric Ph-2/2HbO at pH 8.5 determined by X-ray diffraction at 2.2 Å resolution (diffraction data collected at Synchrotron ESRF, Grenoble) indicates the presence of a water molecule bound to the heme iron atom. This distal water molecule forms a network of hydrogen bonds in the distal cavity, involving Tyr26, Trp93 and another water molecule. The fold is typical of 2/2 Hbs, where the α-helical sandwich is formed by B-E and G-H helices. Moreover, as observed in other group II 2/2 Hbs, the presence of a tunnel connecting the external surface with the protein distal site, is restricted to a few cavities.