(A) Globin structural alignment with Pgbs. Secondary structure elements in globins with known 3D structures are underlined by color and labeled A through H whereas helices predicted by jpred are underlined without color. The pre-A helix is labeled Z, according to the HemAT-Bs structure. Positions at least 50% identical are indicated by bold characters, with the following notations: dagger (†), proximal F8 histidine; pound sign (#), E19 cysteine; psi (ψ), B10 Tyr. Color-coded amino acids are based on an 80% consensus sequence, and colored highlights are assigned to amino acid groups as follows: polar (p, KRHEDQNST) in red; turn-like (t, ACDEGHKNPQRST) in green; bulky hydrophobic (h, ACLIVMHYFW), and aliphatic (l, LIVM) in yellow; aromatic (a, FHWY) in white on pink background; small (s, ACDGNPSTV) in purple; and tiny (u, AGS) in white on purple background. The following sequences and structures were used (PDB IDs in parentheses): HumanMb (5MBN); HHMb (1WLA); SWMb (1MBO); AeFHb (1CQX); Hmp (1GVH); VHb (1VHB); rLHb (1D8U); LHb (2GDM); LpHb (1EBT); GdHb (1VRF); ClNHb (1KR7); PtrHb (1DLW); HbN (1IDR); PCC6803Hb (1MWB); PeHb (1H97); and AscarisHb (1ASH). (B) 3D homology model of ApPgb including the proximal histidine (dark green) and heme (red). The helices are labeled A through H with the disulfide bridge (yellow) indicated with an arrow. MouseHbA, -HbB, -HbE, -Cyg, and -Ngb stand for mouse Hb α, mouse Hb β, mouse Hb ε, mouse cytoglobin, and mouse neuroglobin. HumanHbA, -HbB, -HbG, -HbD, -HbE, -HbT, -Cyg, and -Ngb stand for human Hb α, human Hb β, human Hb γ, human Hb δ, human Hb ε, human Hb θ1, human cytoglobin, and human neuroglobin.

Absorption spectra of ApPgb in different redox states and bound to various ligands. (A) Ferrous (Fe²⁺) ApPgb in the CO-bound (dotted line) and the O₂-bound (dashed and dotted line) forms. (B) Ferric (Fe³⁺) met (solid line) and azido (N₃^-, dashed line) ApPgb. (C) Cyano (CN^-, dotted line) ApPgb. (D) Imidazole-bound (dashed and dotted line) ApPgb. (E) Ferrous deoxy (dotted line) ApPgb saturated with NO to give the Fe²⁺-NO (solid line) species. (F) Ferric met (dotted line) and the Fe³⁺-NO (solid line) species. When NO is added to ferrous deoxy ApPgb in approximately a 20:1 ratio, a spectrum identical to the Fe³⁺-NO spectrum results. All ligands were added to saturation. MaPgb absorption spectra are virtually identical except for an ≈2-nm blue shift at each λ_max. (G) Comparison of wild-type ApPgb bound with imidazole (solid line) to the H120A mutant in the presence of imidazole (dashed and dotted line). The H121A mutant possesses a wild-type spectrum (data not shown). All measurements were done at 25°C and pH 7.0 in either 100 or 200 mM sodium phosphate buffer.

Globin amino acid neighbor-joining phylogenetic tree based on the alignment in Fig. 1. Bootstraps ≥5,000 are indicated at each node. The horizontal scale bar represents 0.1 substitution per site. For abbreviations, see Fig. 1 legend.

Electrophoresis of purified ApPgb and MaPgb. Lane 1, protein molecular weight markers; lane 2, ApPgb_6x-His from A. pernix; lane 3, MaPgb-MBP fusion from M. acetivorans; lane 4, MaPgb-MBP digested with Factor Xa; lane 5, purified MaPgb. The apparently larger molecular weight of ApPgb_6x-His is attributed to the His tag construction.

Parameters for binding of ligands to ferrous ApPgb at 25°C and pH 7.0. (A) Association rates for binding of CO (triangles) and NO (circles), as determined by laser-flash photolysis. (B) Dissociation rate of CO, as determined by oxidation of CO-saturated protein with potassium ferricyanide. (C) Autoxidation of ApPgb on exposure of the deoxy form to air. Details are in Materials and Methods.