Archaeal protoglobin structure suggests novel ligand diffusion paths and heme-reactivity modulation

INTRODUCTION: Despite its strict anaerobic nature, M. acetivorans genome hosts genes that can be related to O2 metabolism; among these, an open reading frame encodes for a “protoglobin” (NP_617780; Pgb). Pgbs are single domain heme proteins of ~195 amino acids, related to the N-terminal domain of archaeal and bacterial globin coupled sensor proteins (GCS) (1-3). Sequence comparisons indicate that Pgbs, despite their 30-35% larger size, are structurally related to the single chain hemoglobins (composed of about 150 amino acids, folded into a 3-on-3 -helical sandwich,12-16% residue identity to Pgbs), and to the heme-based aerotaxis transducer sensor domain of Bacillus subtilis GCS. Pgbs bind O2, CO, and NO reversibly in vitro. Although functional and evolutionary issues are openly debated (1-3), Pgb may facilitate O2 detoxification in vivo promoting electron transfer to O2, or may act as CO sensor/supplier in methanogenesis. RESULTS: We report here the 1.3 Å crystal structure of oxygenated M. acetivorans protoglobin, together with first insight into its ligand binding properties (4). We show that Pgbs are composed of a single heme-binding domain strongly related in tertiary and quaternary structure to the N-terminal domain of archaeal and bacterial GCSs. Furthermore, contrary to all known globins, protoglobin-specific loops and a N-terminal extension completely bury the heme within the protein matrix. Structural modulation of the globin fold in Pgb translates into entirely new access routes to the heme, which is granted by protoglobin-specific apolar tunnels reaching the heme distal site from locations at the B/G and B/E helix interfaces. From a functional viewpoint dimeric protoglobin displays a strikingly modified O2/CO selectivity ratio that favours O2 ligation, and anti-cooperativity in ligand binding. Both properties are exceptional within the hemoglobin superfamily.