Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: Spectral evidence for allosteric regulation by pH and bicarbonate ion

Cobalt(II)-substituted Haemophilus influenzae β-carbonic anhydrase (HICA) has been produced by overexpression in minimal media supplemented with CoCl2, enabling kinetic, structural, and spectroscopic characterization. Co(II)-substituted HICA (Co-HICA) has comparable catalytic activity to that of wild-type enzyme with kcat = 82 ± 19 ms−1 (120% of wild-type). The X-ray crystal structure of Co-HICA was determined to 2.5 Å resolution, and is similar to the zinc enzyme. The absorption spectrum of Co-HICA is consistent with four-coordinate geometry. pH-dependent changes in the absorption spectrum of Co-HICA, including an increase in molar absorptivity and a red shift of a 580 nm peak with decreasing pH, correlate with the pH dependence of kcat/Km. The absence of isosbestic points in the pH-dependent absorption spectra suggest that more than two absorbing species are present. The addition of bicarbonate ion at pH 8.0 triggers spectral changes in the metal coordination sphere that mimic that of lowering pH, supporting its hypothesized role as an allosteric inhibitor of HICA. Homogeneously (99 ± 1% Co) and heterogeneously (52 ± 5% Co) substituted Co-HICA have distinctly different colors and absorption spectra, suggesting that the metal ions in the active sites in the allosteric dimer of Co-HICA engage in intersubunit communication.

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► A simple method for producing Co(II)-substituted Haemophilus influenza β-carbonic anhydrase (Co-HICA) is described.
► Cobalt substitution provides a direct metric of the hypothesized cooperativity of HICA.
► Lack of isosbestic points in the pH-dependent absorption spectra suggests more than two absorbing species in the active site.
► Bicarbonate ion at pH 8.0 triggers spectral changes in the metal coordination sphere mimicking that of lowering pH.
► Heterogeneously substituted Co-HICA has a different color and absorption spectrum, suggesting intersubunit communication.