Crystal structure of heme A synthase from Bacillus subtilis

Abstract

Heme A is an essential cofactor for respiratory terminal oxidases and vital for respiration in aerobic organisms. The final step of heme A biosynthesis is formylation of the C-8 methyl group of heme molecule by heme A synthase (HAS). HAS is a heme-containing integral membrane protein, and its structure and reaction mechanisms have remained unknown. Thus, little is known about HAS despite of its importance. Here we report the crystal structure of HAS from Bacillus subtilis at 2.2-Å resolution. The N- and C-terminal halves of HAS consist of four-helix bundles and they align in a pseudo twofold symmetry manner. Each bundle contains a pair of histidine residues and forms a heme-binding domain. The C-half domain binds a cofactor-heme molecule, while the N-half domain is vacant. Many water molecules are found in the transmembrane region and around the substrate-binding site, and some of them interact with the main chain of transmembrane helix. Comparison of these two domain structures enables us to construct a substrate-heme binding state structure. This structure implies that a completely conserved glutamate, Glu57 in B. subtilis, is the catalytic residue for the formylation reaction. These results provide valuable suggestions of the substrate-heme binding mechanism. Our results present significant insight into the heme A biosynthesis.

Keywords: CtaA; crystal structure; formylation reaction; heme A biosynthesis; membrane protein.

Fig. 1.

Overall structure of BsHAS. (A) Scheme of heme A biosynthesis. The hydroxyethylfarnesyl group and the formyl group are shaded in green. (B) Overall structure of BsHAS. The structure is a ribbon drawing as viewed from within the membrane and the extracellular side. The N-half domain is shown in cyan and C-half is shown in pink. The heme molecule is shown as spheres. (C) Heme-binding site. A section of the surface representation of BsHAS is shown.

Fig. 2.

Pseudo twofold symmetric structure. (A) Structural superimposition of transmembrane helices of the N (cyan)- and C (pink)-half domains. (B) The helical secondary structure breaking region of TM2. The hydrogen bonds in the main chain are indicated by black dashes and the others are shown with green dashes. (C) TM4. The omit map for waters is shown in cyan (4.0σ).

Fig. 3.

Cofactor-heme binding site. (A) Omit map of the cofactor-heme for determination of its orientation and conformation. The heme b is viewed from the α-face (back side). The omit map is shown for the whole heme molecule (gray mesh, 1.5σ; red mesh, 10.0σ). (B) Environment of the cofactor-heme b. The 2mF_obs − DF_calc electron density map is shown (gray mesh, 1.0σ; magenta mesh, 8.0σ). (C) Hydrogen bond network. Water molecules are shown as red spheres with an omit map in cyan (4.0σ), and amino acid residues included in the hydrogen bond network are shown as sticks. The dotted lines indicate hydrogen bonds.

Fig. 4.

Substrate-heme binding site. (A) Position of ECL1 and transmembrane helices. The molecular surface of ECL1 is colored cyan. (B) Interaction in the substrate-heme binding site. The ECL1 region is shaded in pale cyan. Glu57 interacts with the main chain of Gly36 in ECL1. (C) A proposed model of the substrate-heme binding state viewed from within the membrane. The heme O molecule is shown as spheres. (D) The catalytic site of the substrate-heme binding state in the proposed model. (E) Substrate heme O binding and conformational changes of ECL1 and TM2.