Journal:JBSD:9

Carbon monoxide binding to the heme group at the dimeric interface modulates structure and copper accessibility in the Cu,Zn superoxide dismutase from Haemophilus ducreyi: in silico and in vitro evidences

Giovanni Chillemi, Serena De Santis, Mattia Falconi, Giordano Mancini, Valentina Migliorati, Andrea Battistoni, Francesca Pacello, Alessandro Desideri, Paola D’Angelo ^[1]

Molecular Tour
Superoxide dismutases (SODs) are metalloenzymes playing a vital role in the defense mechanism against the oxidative stress; they catalyze the dismutation of superoxide, the one-electron reduction product of oxygen, to hydrogen peroxide and molecular oxygen, thus protecting living organism from oxidative lethality (conserved SODs regions among prokaryotes and eukaryotic organisms are highlighted in different colors: SS subloop (residues Glu73-Gly93) in crimson; (Zn subloop (residues Gly94-Ala119) in salmon; (Greek key loop (residues Pro135-Gly145) in violet color; (7,8 loop (residues Ala152-Pro169) in pink color). Haemophilus ducreyi, the causative agent of the sexually transmitted human genital ulcerative disease known as chancroid, expresses one of the most interesting examples of bacterial Cu,Zn SOD (#HdSOD) with the unique feature of binding a heme molecule at the interface between the two subunits asymmetrically bound by residues His64 and His124 of subunits A and B, respectively (His64 and His124 are colored green and heme molecule is in yellow). The heme molecule proved to be able to bind small gaseous ligands, such as nitric oxide or carbon monoxide, as a sixth ligand thus displacing the distal histidine. In this study the structural and dynamic response of HdSOD to the binding of CO to heme (the carbon atom of CO colored in magenta and the oxigen one is in red) was studied by means of a combinations of Molecular Dynamics Simulation and X-Ray absorbtion spectroscopy and hypothesis formulated were further confirmed by in vitro experiments. All together the collected results evidenced that binding of the CO molecule produces a strong reduction in the asymmetric fluctuations of the two subunits and long range effects of the heme group on the Cu active site structure (active site residues: His70, His72, His95, His104, His113, Asp 116 and His 151 are in cyan, Zn is in gray and Cu is in darkgoldenrod) which becomes more easily accessible to the solvent thus causing an increase in copper dismutasic activity. Based on this picture, we suggest a role of HdSOD as a heme-based-sensor protein in which conformational changes, triggered by the heme group, could help Haemophilus ducreyi to adapt at fluctuating levels of gaseous molecules, such as carbon monoxide or nitric oxide: in the presence of these gasses, the HdSOD would be able to increase its superoxide dismutation activity to subtract superoxide substrate and to prevent the formation of the dangerous peroxynitrite, the molecule generated by the reaction of nitric oxide with superoxide, which is even more harmful to the cell than superoxide. Further investigation to validate this attractive hypothesis is thus desirable.