The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

Abstract

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 a-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.

Structural flexibility of the heme cavity in the cold‐adapted truncated hemoglobin from the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125

“…4A), characterized by a Soret band at 408 nm, Q bands at 503, 541 and 570 nm, and a CT1 band at 635 nm, suggest a mixture of high-spin (HS) and lowspin (LS) forms. This is confirmed by the corresponding high-frequency RR spectrum ( for ferric Ph-2/2HbO at pH 7.6 [13] where, probably due to different preparation procedures, multiple LS forms were observed (see Materials and methods). However, as reported in that case, the absorption maxima of the LS form are quite unusual, and reminiscent of those of ferric Chlamydomonas chloroplast Hb [23] and of the hemophore HasA proteins from Serratia marcescens and Pseudomonas aeruginosa [24,25].…”

“…Analysis of the crystal structure reveals that in Ph-2/ 2HbO the CD1 topological position is occupied by His58, not by Tyr55 as previously proposed [13]. CD1 His is also present in At-2/2HbO, whereas in TrHbs this site typically hosts Phe, with the exception of TrHbIIs from M. tuberculosis, Mycobacterium avium, Mycobacterium leprae, Mycobacterium smegmatis, Streptomyces coelicolor, Corynebacterium diphtheriae and T. fusca, which host Tyr [5,20].…”

“…The previously used fermentor [13] was replaced by small-scale (200 mL) cultures in shake flasks to improve the quality of expression. In fact, production of the protein under strong oxygenation conditions may have given rise to the multiple LS forms observed previously [13].…”

“…The previously used fermentor [13] was replaced by small-scale (200 mL) cultures in shake flasks to improve the quality of expression. In fact, production of the protein under strong oxygenation conditions may have given rise to the multiple LS forms observed previously [13]. For overexpression of Ph-2/2HbO in E. coli BL21(DE3), a single colony carrying the plasmid construct (pET28a-Ph-2/2HbO) was inoculated in LB medium supplemented with kanamycin (50 lgÁmL À1 ) in the presence of 0.3 mM D-aminolevulinic acid and allowed to grow at 25°C until A 600 reached 0.6.…”

“…In particular, TrHbII encoded by the PSHAa0030 gene, named Ph-2/2HbO, has been thoroughly investigated from the biochemical and functional viewpoints [10,[12][13][14][15][16]. The inactivation of the PSHAa0030 gene encoding Ph-2/2HbO makes the mutant bacterial strain sensitive to high O 2 levels, hydrogen peroxide and nitrosating agents [12].…”