Crystal structure and biochemical features of EfeB/YcdB from Escherichia coli O157: ASP235 plays divergent roles in different enzyme-catalyzed processes

Abstract

EfeB/YcdB is a member of the dye-decolorizing peroxidase (DyP) protein family. A recent study has shown that this protein can extract iron from heme without breaking the tetrapyrrole ring. We report the crystal structure of EfeB from Escherichia coli O157 bound to heme at 1.95 Å resolution. The EfeB monomer contains two domains. The heme molecule is located in a large hydrophobic pocket in the C-terminal domain. A long loop connecting the two domains extensively interacts with the heme, which is a distinctive structural feature of EfeB homologues. A large tunnel formed by this loop and the β-sheet of C-terminal domain provides a potential cofactor/substrate binding site. Biochemical data show that the production of protoporphyrin IX (PPIX) is closely related to the peroxidation activity. The mutant D235N keeps nearly the same activity of guaiacol peroxidase as the wild-type protein, whereas the corresponding mutation in the classic DyP protein family completely abolished the peroxidation activity. These results suggest that EfeB is a unique member of the DyP protein family. In addition, dramatically enhanced fluorescence excitation and emission of EfeB-PPIX was observed, implying this protein may be used as a red color fluorescence marker.

FIGURE 1.

Fluorescence characteristic of free PPIX and EfeB-PPIX complex. The initial concentration of PPIX is 2 mm and was diluted to different concentrations from 10 to 60 μm using Tris-HCl buffer (10 mm Tris-HCl, pH 8.0, 100 mm NaCl). Apo-EfeB was diluted to different concentrations from 10 to 60 μm using the same buffer, and then PPIX was added to the apo-EfeB solution to a final concentration ranging from 10 to 60 μm and was mixed and measured. A, excitation and emission wavelength scan for free PPIX of 10 μm, showing that the distinctive excitation wavelength is 398 nm and emission wavelength is 620 nm. B, excitation and emission wavelength scan for EfeB-PPIX complex of 10 μm, showing that the excitation wavelength is 407.5 nm and emission wavelength is 622.5 nm. C, fluorescence intensity of free PPIX increased nonlinearly as concentration rises. However, the fluorescence intensity of EfeB-PPIX complex increased linearly with rising concentrations.

FIGURE 2.

Stereoview of the structure of heme-bound EfeB monomer and electron density for the heme moiety. A, rainbow-colored scheme representation of the EfeB-heme complex. 12 α-helices and 8 β-strands are labeled. The heme molecule is shown as a stick. The S loop represents a switch loop and is colored in magenta. B, difference electron density map (F_o-F_c) calculated at 1.95 Å resolution using phase from the final model with heme and waters omitted and contoured at 1.5σ reveals an electron density consistent with the heme molecule within molecule A.

FIGURE 3.

Structure of the homodimer and stereoview of the dimer interface. The homodimer is shown in schematic representation; molecule A is in blue and molecule B is in green. Two heme molecules are shown in stick representation. The zoomed region shows the secondary structures contributing to the dimer formation. The residues that form the interface are shown in sticks with molecule A in blue and molecule B in green.

FIGURE 4.

Stereoview of heme binding site. The heme carbons are yellow sticks with iron atoms shown as brown spheres. Residues contributing to heme binding are shown as sticks with carbons in gray. The red stick represents the oxygen molecule and acts as the distal ligand. The water molecules are depicted as violet-purple spheres. The hydrogen bonds are depicted as dashed lines.

FIGURE 5.

PPIX production of EfeB mutants tested by fluorescence scan of cell lysates of bacteria overexpressing mutants. Native EfeB was used as a positive control. A, excitation wavelength scan for mutants and native EfeB. The obvious excitation wavelength at 407.5 nm was detected for EfeB. No distinctive excitation wavelength was observed for these mutants except for D235N,D235A and S351Q, which kept about 102, 17, and 8% fluorescence intensity of native EfeB, respectively. B, emission wavelength scan for mutants and native EfeB. The distinct emission wavelength at 622.5 nm was observed for EfeB, no emission wavelength was detected for these mutants except for D235N,D235A and S351Q, which kept about 102, 17, and 8% fluorescence intensity of native EfeB, respectively.

FIGURE 6.

The unidentified cofactor binding tunnel. The protein is shown in surface representation with a cross-sectioned slab removed to allow viewing of the internal ligand-binding tunnel. 26 residues involved in the formation of the tunnel are shown as sticks with carbons in orange.

FIGURE 7.

Sequence alignment of EfeB and its homologous proteins. Residues that make up the tunnel are labeled with solid squares at the top, and residues involved in stabilization of heme molecule are marked in solid triangles at the bottom. The proximal ligand His³²⁹ is in dark gray and the switch loop is highlighted using a gray line.

FIGURE 8.

Correlation of peroxidase activity and the PPIX production. Both guaiacol and catechol were used as substrates to determine the peroxidase activities of EfeB and its mutants. PPIX production was measured using fluorescence spectroscopy. The peroxidase activity and PPIX production of native EfeB were set to 100%.