Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

Abstract

Despite the large number of globins recently discovered in bacteria, our knowledge of their physiological functions is restricted to only a few examples. In the microbial world, globins appear to perform multiple roles in addition to the reversible binding of oxygen; all these functions are attributable to the heme pocket that dominates functional properties. Resistance to nitrosative stress and involvement in oxygen chemistry seem to be the most prevalent functions for bacterial globins, although the number of globins for which functional roles have been studied via mutation and genetic complementation is very limited. The acquisition of structural information has considerably outpaced the physiological and molecular characterisation of these proteins. The genome of the Antarctic cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 (PhTAC125) contains genes encoding three distinct single-chain 2/2 globins, supporting the hypothesis of their crucial involvement in a number of functions, including protection against oxidative and nitrosative stress in the cold and O-rich environment. In the genome of PhTAC125, the genes encoding 2/2 globins are constitutively transcribed, thus suggesting that these globins are not functionally redundant in their physiological function in PhTAC125. In the present study, the physiological role of one of the 2/2 globins, Ph-2/2HbO-2217, was investigated by integrating in vivo and in vitro results. This role includes the involvement in the detoxification of reactive nitrogen and O species including NO by developing two in vivo and in vitro models to highlight the protective role of Ph-2/2HbO-2217 against reactive nitrogen species. The PSHAa2217 gene was cloned and over-expressed in the flavohemoglobin-deficient mutant of Escherichia coli and the growth properties and O uptake in the presence of NO of the mutant carrying the PSHAa2217 gene were analysed. The ferric form of Ph-2/2HbO-2217 is able to catalyse peroxynitrite isomerisation in vitro, indicating its potential role in the scavenging of reactive nitrogen species. Here we present in vitro evidence for the detoxification of NO by Ph-2/2HbO-2217.

Structural determinants of ligand binding in truncated hemoglobins: Resonance Raman spectroscopy of the native states and their carbon monoxide and hydroxide complexes

“…EPR and RR spectra have confirmed the presence of a tyrosinate coordinated to the heme iron . Figure shows the RR spectra of Ph ‐HbO‐0030 at pH 7.6, obtained with excitation at 514.5 nm, that is, in resonance with the tyrosinate–Fe(III) charge transfer band (near 500 nm) .…”

“…Our group has worked extensively on representative proteins of HbII (or HbO), namely Thermobifida fusca ( Tf ‐HbO) and Pseudoalteromonas haloplanktis TAC125 ( Ph ‐HbOs) . While the former Hb is obtained from a thermophilic actinobacterium, T. fusca, the other proteins are from the cold‐adapted bacterium P. haloplanktis TAC125 which contains genes encoding three distinct 2/2Hbs …”

“…Figure shows the sequence alignments of Ph ‐2/2HbO‐2217, Ph ‐2/2HbO‐0030, and Tf‐ HbO . The sequence identity between the two Antarctic globins is only 24%, whereas it is 33 and 27% between T. fusca and Ph ‐2/2HbO‐0030, and Ph ‐2/2HbO‐2217, respectively.…”

“… Sequence alignments, carried out by Clustal Omega, of Ph‐ 2/2HbO‐2217, Ph‐ 2/2HbO‐0030 and Tf ‐HbO . Manual adjustments have been based on known crystal structures adapted from .…”

“…The structure of Ph ‐HbO‐2217 has not been yet solved; however, recently the Ph ‐2/2HbO‐0030 structure has been used as template to obtain the Ph ‐HbO‐2217 homology model . In this regard, it is important to note that although the use of structural templates can be a valuable resource in the absence of the crystallographic structure of a protein, its limitations should be borne in mind and the significance of the inferences drawn viewed with the due caution.…”