Carbonic anhydrases catalyze the reversible hydration of CO ₂ and are ubiquitous in highly evolved eukaryotes. The recent identification of a third class of carbonic anhydrase (γ class) in a methanoarchaeon and our present finding that the β class also extends into thermophilic species from the Archaea domain led us to initiate a systematic search for these enzymes in metabolically and phylogenetically diverse prokaryotes. Here we show that carbonic anhydrase is widespread in the Archaea and Bacteria domains, and is an ancient enzyme. The occurrence in chemolithoautotrophic species occupying deep branches of the universal phylogenetic tree suggests a role for this enzyme in the proposed autotrophic origin of life. The presence of the β and γ classes in metabolically diverse species spanning the Archaea and Bacteria domains demonstrates that carbonic anhydrases have a far more extensive and fundamental role in prokaryotic biology than previously recognized.

The conversion of biomass to CH4 (biomethanation) involves an anaerobic microbial food chain composed of at least three metabolic groups of which the first two decompose the complex biomass primarily to acetate, formate, and H2. The thermodynamics of these conversions are unfavorable requiring a symbiosis with the CH4-producing group (methanogens) that metabolize the decomposition products to favorable concentrations. The methanogens produce CH4 by two major pathways, conversion of the methyl group of acetate and reduction of CO2 coupled to the oxidation of formate or H2. This review covers recent advances in the fundamental understanding of both methanogenic pathways with the view of stimulating research towards improving the rate and reliability of the overall biomethanation process.

Methane produced in the biosphere is derived from two major pathways. Conversion of the methyl group of acetate to CH₄ in the aceticlastic pathway accounts for at least two-thirds, and reduction of CO₂ with electrons derived from H₂, formate, or CO accounts for approximately one-third. Although both pathways have terminal steps in common, they diverge considerably in the initial steps and energy conservation mechanisms. Steps and enzymes unique to the CO₂ reduction pathway are confined to methanogens and the domain Archaea. On the other hand, steps and enzymes unique to the aceticlastic pathway are widely distributed in the domain Bacteria, the understanding of which has contributed to a broader understanding of prokaryotic biology.

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (α class) and plants (β class); however, two additional classes (γ and δ) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the γ class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo-Cam with Fe²⁺, which yielded Cam with an effective k_cat that exceeded that for the Zn²⁺-reconstituted enzyme. Mössbauer spectroscopy showed that the Fe²⁺-reconstituted enzyme contained high spin Fe²⁺ that, when oxidized to Fe³⁺, inactivated the enzyme. Reconstitution with Fe³⁺ was unsuccessful. Reconstitution with Cu²⁺, Mn²⁺, Ni²⁺, or Cd²⁺ yielded enzymes with effective k_cat values that were 10% or less than the value for the Zn²⁺-reconstituted Cam. Cam produced in Escherichia coli and purified anaerobically contained iron with effective k_cat and k_cat/K_m values exceeding the values for Zn²⁺-reconstituted Cam. The results identify a previously unrecognized biological function for iron.