letters to the editor
Comment on the article Structure and mechanism of copper–carbonic anhydrase II: a nitrite reductase
aInstitute of Toxicology, Core Unit Proteomics, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover, 30625, Germany
*Correspondence e-mail: tsikas.dimitros@mh-hannover.de
Keywords: carbonic anhydrase; nitrite reductase; nitrous anhydrase; protein structure; enzyme mechanisms; inorganic chemistry.
Carbonic anhydrase (CA) is one of the oldest, most efficient and best investigated ubiquitous Zn2+-containing enzymes. CA catalyzes a very simple but vital reaction, i.e. the hydration of carbon dioxide, in mammals, plants and bacteria (Meldrum & Roughton, 1933). Rather surprisingly, over recent decades many additional physiological and pathological roles of CA have been discovered. A newly discovered CA activity is the bioactivation of inorganic nitrite (O=N—O−) to nitric oxide (NO), a signaling multiple-functional gaseous molecule in living organisms. Central to scientific research on CA has been its catalytic site that preferentially binds Zn2+, which is redox-inactive, and Cu2+, which is redox-active (Lindskog & Nyman, 1964; Coleman, 1965). This topic is still of great scientific interest (Kim et al., 2020). In addition, and in contrast to Zn2+, Cu2+ binds to two different centers of CA which are differently affected by glutathione (GSH), the most abundant endogenous intra-cellular antioxidant with high specificity to Zn2+, Cu2+ and other divalent ions including Hg2+ (Tabbì et al., 2019). It can be expected that Cu2+-carrying CA is likely to exert not only the classical carbonic anhydrase activity, but may also be involved in redox-dependent reactions and mechanisms. For example, Cu2+-containing CA could oxidize NO to nitrite and higher nitrogen oxides (NOx), as performed by the Cu2+-rich ceruloplasmin, or it could reduce nitrite to NO via intermediate Cu+-formation by GSH or ascorbic acid (Tabbì et al., 2019). Such a reaction is practically impossible for regular Zn2+-containing CA.
Recently, Andring and associates reported the 2+-hCAII) in complex with inorganic nitrite (O=N—O−) at 1.2 Å resolution with two Cu2+ centers, analogous to bacterial nitrite reductases, and suggested that Cu2+-hCAII can function as a nitrite reductase, yet without providing experimental evidence (Andring et al., 2020). In the scientific commentary on this article, Liljas stated that `Andring et al. (2020) have been able to unravel the mystery' (Liljas, 2020), probably referring to the controversy that Aamand et al. (2009) found Zn2+-CAII to reduce nitrite to NO, whereas Andring et al. (2018) failed to detect Zn2+-CAII-mediated reduction of nitrite to NO.
of copper (II)-bound human carbonic anhydrase II (CuOur studies using bovine and human Zn2+-CAII demonstrated formation of S-nitroso-glutathione (GSNO) from nitrite and GSH suggesting nitrous anhydrase activity of Zn2+-CAII, which was not inhibitable by the CA-inhibitors acetazolamide or dorzolamide (Hanff et al., 2016; Zinke et al., 2016). We observed formation of NO only in the presence of L-cysteine (CysSH), most likely due to the intermediate formation of S-nitrosocysteine (CysSNO), which can readily and abundantly decompose to NO in the presence of Cu+ (Tsikas et al., 2002).
Cu2+ ions were found to bind to Zn2+-CAII isolated from human erythrocytes at a site other than the active site and inhibited the exchange of water from the enzyme without affecting the equilibrium rate of hydration of CO2 (Tu et al., 1981). This observation may suggest that classical CA inhibitors such as acetazolamide may inhibit the carbonic anhydrase activity of CA by tightly binding to the CAII-bound Zn2+, through the sulfone amide group, but not to the second Cu2+-binding site. This could be an explanation for our observation that neither acetazolamide nor dorzolamide inhibited the nitrous anhydrase activity of bovine and human CAII (Hanff et al., 2016; Zinke et al., 2016).
Andring et al. (2020) stated that `recent reports have shown that CAII can also reduce nitrite (NO2−) to nitric oxide (NO)... (Andring et al., 2018; Aamand et al., 2009; Hanff et al., 2018)', that `However, when dialyzed with ethylenediaminetetraacetic acid (EDTA), the enzyme retained its carbonic anhydrase activity yet lost its nitrite reductase activity (Hanff et al., 2018)', and that `Furthermore, if this bovine CAII was dialyzed against EDTA, the nitrite reductase activity was ablated indicating that a metal cofactor within the bovine blood was needed for the CAII-dependent nitrite reductase activity (Andring et al., 2018; Hanff et al., 2018).'. We wish to point out this mistake in the paper by Andring et al. (2020). In the paper referred to above (i.e., Hanff et al., 2018), we did not report that CAII is a nitrite reductase, but we explicitly stated that we measured nitrous anhydrase activity of bovine and human CAII and CAIV, and did not use EDTA (i.e. Hanff et al., 2018).
References
Aamand, R., Dalsgaard, T., Jensen, F. B., Simonsen, U., Roepstorff, A. & Fago, A. (2009). Am. J. Physiol. Heart Circ. Physiol. 297, H2068–H2074. Web of Science CrossRef PubMed CAS Google Scholar
Andring, J. T., Kim, C. U. & McKenna, R. (2020). IUCrJ, 7, 287–293. Web of Science CrossRef CAS PubMed IUCr Journals Google Scholar
Andring, J. T., Lomelino, C. L., Tu, C., Silverman, D. N., McKenna, R. & Swenson, E. R. (2018). Free Radical Biol. Med. 117, 1–5. Web of Science CrossRef CAS Google Scholar
Coleman, J. E. (1965). Biochemistry, 4, 2644–2655. CrossRef CAS PubMed Web of Science Google Scholar
Hanff, E., Böhmer, A., Zinke, M., Gambaryan, S., Schwarz, A., Supuran, C.T. & Tsikas, D. (2016). Amino Acids, 48, 1695–706. Web of Science CrossRef CAS PubMed Google Scholar
Hanff, E., Zinke, M., Böhmer, A., Niebuhr, J., Maassen, M., Endeward, V., Maassen, N., & Tsikas, D. (2018). Anal. Biochem. 550, 132–136. Web of Science CrossRef CAS PubMed Google Scholar
Kim, J. K., Lee, C., Lim, S. W., Adhikari, A., Andring, J. T., McKenna, R., Ghim, C.-M. & Kim, C. U. (2020). Nat. Commun. 11, 4557. Web of Science CrossRef PubMed Google Scholar
Liljas, A. (2020). IUCrJ, 7, 144–145. Web of Science CrossRef CAS PubMed IUCr Journals Google Scholar
Lindskog, S. & Nyman, P. O. (1964). Biochim. Biophys. Acta, 85, 462–474. PubMed CAS Web of Science Google Scholar
Meldrum, N. U. & Roughton, F. J. (1933). J. Physiol. 80, 113–142. CrossRef PubMed CAS Google Scholar
Tabbì, G., Magrì, A. & Rizzarelli, E. (2019). J. Inorg. Biochem. 199, 110759. Web of Science PubMed Google Scholar
Tsikas, D., Sandmann, J. & Frölich, J. C. (2002). J. Chromatogr. B, 772, 335–346. Web of Science CrossRef CAS Google Scholar
Tu, C., Wynns, G. C. & Silverman, D. N. (1981). J. Biol. Chem. 256, 9466–9470. CrossRef CAS PubMed Google Scholar
Zinke, M., Hanff, E., Böhmer, A., Supuran, C. T. & Tsikas, D. (2016). Amino Acids, 48, 245–255. Web of Science CrossRef CAS PubMed Google Scholar
This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.