A role for iron in an ancient carbonic anhydrase.
Since 1933, carbonic anhydrase research has focused on enzymes from mammals (alpha class) and plants (beta class); however, two additional classes (gamma and delta) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the gamma 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 kcat 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 kcat 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 kcat and kcat/Km values exceeding the values for Zn2+-reconstituted Cam. The results identify a previously unrecognized biological function for iron.
Tripp
Brian C
BC
Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, Pennsylvania 16802-4500, USA.
Bell
Caleb B
CB
Cruz
Francisco
F
Krebs
Carsten
C
Ferry
James G
JG
D001105:Archaea / Q000378:metabolism; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D017020:Methanosarcina / Q000378:metabolism; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D015032:Zinc / Q000737:chemistry
D002104:Cadmium; D006861:Hydrogen Peroxide; D007501:Iron; D002256:Carbonic Anhydrases; D015032:Zinc