The Archetype γ-Class Carbonic Anhydrase (Cam) Contains Iron When Synthesized in Vivo

MacAuley, Sheridan R.; Zimmerman, Sabrina A.; Apolinario, Ethel E.; Evilia, Caryn; Hou, Ya‐Ming; Ferry, James G.; Sowers, Kevin R.

doi:10.1021/bi802246s

Cited by 81 publications

(58 citation statements)

References 16 publications

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“…We found that iron is the more catalytically active metal for Mt-CamH, and this enzyme is only the second CA shown to utilize this metal for catalysis. Mt-Cam contains Fe 2ϩ in the active site when it is overproduced in the closely related species M. acetivorans (18), a result that establishes that iron is the physiologically relevant metal in Mt-Cam and also supports the hypothesis that iron is the physiologically relevant metal in Mt-CamH.…”

Section: Discussionsupporting

confidence: 62%

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Characterization of CamH from Methanosarcina thermophila , Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

2010

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The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the ␥ class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from M. thermophila (Mt-CamH) was overproduced in Escherichia coli and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from E. coli cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had k cat and k cat /K m values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from E. coli cultured with supplemental iron (Fe-MtCamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective k cat (k cat eff ) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the k cat /K m eff was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the k cat eff of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H 2 O 2 , which supports the hypothesis that Fe 2؉ has a role in the active site. The k cat for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a "ping-pong" mechanism accepting a proton. The k cat /K m was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO 2 and HCO 3 ؊ is separate from intermolecular proton transfer.

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Section: Discussionsupporting

confidence: 62%

“…Mt-Cam overproduced in Escherichia coli and purified aerobically contains zinc in the active site (2). However, overproduction in the closely related species Methanosarcina acetivorans yields an enzyme with 3-fold-greater CA activity and iron in the active site, establishing iron as the physiologically relevant metal (18).…”

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confidence: 99%

Characterization of CamH from Methanosarcina thermophila , Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

2010

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“…Because it is highly unlikely that CO can bind directly to the implicated amino acid residues (His 365 , His 394 , and Asp 367 ) in the RCK1 domain in a metal-independent manner, one possibility is that the His and Asp residues in RCK1 ligate a lowvalent metal ion, e.g. (52). Thus, further studies are required to clarify the relationship between CO and the potential metal binding sites in the RCK1 domain.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Morgan

Ragsdale

2010

Journal of Biological Chemistry

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Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large- RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K d ‫؍‬ 210 nM) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K d ‫؍‬ 50 nM) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.

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“…The different results obtained in aerobic and anaerobic conditions is explained by the fact that in aerobic conditions Fe 3+ is oxidized and rapidly loss from CAM enzyme, substituted by Zn 2+ contaminating buffers not treated with chelating agents. These results indicate Fe 2+ as the physiologically relevant metal [MacAuley et al, 2009;Tripp et al, 2004] in the active site for CAM enzyme. Interestingly, evidence for the role of ferrous ion in CA has been obtained also for the class.…”

Section: Heavy Metals As Carbonic Anhydrase Cofactorsmentioning

confidence: 62%

Carbonic Anhydrase and Heavy Metals

Lionetto¹,

Caricato²,

Giordano³

et al. 2012

Biochemistry

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The Archetype γ-Class Carbonic Anhydrase (Cam) Contains Iron When Synthesized in Vivo

Cited by 81 publications

References 16 publications

Characterization of CamH from Methanosarcina thermophila , Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

Characterization of CamH from Methanosarcina thermophila , Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Carbonic Anhydrase and Heavy Metals

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