Carbon Dioxide "Trapped" in a β-Carbonic Anhydrase

Abstract

Carbonic anhydrases (CAs) are enzymes that catalyze the hydration/dehydration of CO2/HCO3(-) with rates approaching diffusion-controlled limits (kcat/KM ∼ 10(8) M(-1) s(-1)). This family of enzymes has evolved disparate protein folds that all perform the same reaction at near catalytic perfection. Presented here is a structural study of a β-CA (psCA3) expressed in Pseudomonas aeruginosa, in complex with CO2, using pressurized cryo-cooled crystallography. The structure has been refined to 1.6 Å resolution with R(cryst) and R(free) values of 17.3 and 19.9%, respectively, and is compared with the α-CA, human CA isoform II (hCA II), the only other CA to have CO2 captured in its active site. Despite the lack of structural similarity between psCA3 and hCA II, the CO2 binding orientation relative to the zinc-bound solvent is identical. In addition, a second CO2 binding site was located at the dimer interface of psCA3. Interestingly, all β-CAs function as dimers or higher-order oligomeric states, and the CO2 bound at the interface may contribute to the allosteric nature of this family of enzymes or may be a convenient alternative binding site as this pocket has been previously shown to be a promiscuous site for a variety of ligands, including bicarbonate, sulfate, and phosphate ions.

Figure 1

Surface representation of (A) hCA II and (B) psCA3. Beige and green regions represent hydrophilic and hydrophobic residues of the active site, respectively. Stick representations of the active site for (C) hCA II and (D) psCA3. The active site zinc is depicted as a magenta sphere (only one active site of the psCA3 dimer is visible in this view). For all subsequent figures, residues will be as labeled, ordered waters depicted as red spheres, zinc depicted as a magenta sphere, H-bonds represented by red dashes, and distances given in angstroms.

Figure 2

Cartoon representation of the functional dimeric form of psCA3. The green mesh is the difference (F_o − F_c) electron density for the two CO₂ binding sites, contoured at 2.0σ. The right panels show details of the CO₂ binding sites. The blue mesh is the refined (2F_o − F_c) electron density after modeling building and refinement, contoured at 1.6σ.

Figure 3

Hydrogen bond network in the psCA3 active site: (A) open (without CO₂) and (B) with CO₂. Residues are as labeled. Beige and green regions represent hydrophilic and hydrophobic residues, respectively. Note the displacement of deep water upon CO₂ binding.

Figure 4

Secondary binding site for another molecule of CO₂ shown at the dimeric interface as (A) a cartoon and (B) sticks (close-up). The two monomers are colored yellow and cyan.

Figure 5

Superposition of unbound psCA3 (gray) and CO₂-bound (green) represented as a (A) cartoon and (B) close-up sticks, showing the slight perturbation in two active site residues.

Figure 6

Cross-eyed stereoview of CO₂ in the active site of (A) hCA II and (B) psCA3. Residues are as labeled.