letters to the editor\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

IUCrJ
Volume 8| Part 2| March 2021| Pages 327-328
ISSN: 2052-2525

Comment on the article Structure and mechanism of copper–carbonic anhydrase II: a nitrite reductase

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aInstitute of Toxicology, Core Unit Proteomics, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover, 30625, Germany
*Correspondence e-mail: tsikas.dimitros@mh-hannover.de

Edited by E. N. Baker, University of Auckland, New Zealand (Received 4 November 2020; accepted 23 December 2020; online 18 January 2021)

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[Meldrum, N. U. & Roughton, F. J. (1933). J. Physiol. 80, 113-142.]). 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[Lindskog, S. & Nyman, P. O. (1964). Biochim. Biophys. Acta, 85, 462-474.]; Coleman, 1965[Coleman, J. E. (1965). Biochemistry, 4, 2644-2655.]). This topic is still of great scientific interest (Kim et al., 2020[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.]). 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[Tabbì, G., Magrì, A. & Rizzarelli, E. (2019). J. Inorg. Biochem. 199, 110759.]). 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[Tabbì, G., Magrì, A. & Rizzarelli, E. (2019). J. Inorg. Biochem. 199, 110759.]). Such a reaction is practically impossible for regular Zn2+-containing CA.

Recently, Andring and associates reported the crystal structure of copper (II)-bound human carbonic anhydrase II (Cu2+-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[Andring, J. T., Kim, C. U. & McKenna, R. (2020). IUCrJ, 7, 287-293.]). In the scientific commentary on this article, Liljas stated that `Andring et al. (2020[Andring, J. T., Kim, C. U. & McKenna, R. (2020). IUCrJ, 7, 287-293.]) have been able to unravel the mystery' (Liljas, 2020[Liljas, A. (2020). IUCrJ, 7, 144-145.]), probably referring to the controversy that Aamand et al. (2009[Aamand, R., Dalsgaard, T., Jensen, F. B., Simonsen, U., Roepstorff, A. & Fago, A. (2009). Am. J. Physiol. Heart Circ. Physiol. 297, H2068-H2074.]) found Zn2+-CAII to reduce nitrite to NO, whereas Andring et al. (2018[Andring, J. T., Lomelino, C. L., Tu, C., Silverman, D. N., McKenna, R. & Swenson, E. R. (2018). Free Radical Biol. Med. 117, 1-5.]) failed to detect Zn2+-CAII-mediated reduction of nitrite to NO.

Our 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[Hanff, E., Böhmer, A., Zinke, M., Gambaryan, S., Schwarz, A., Supuran, C.T. & Tsikas, D. (2016). Amino Acids, 48, 1695-706.]; Zinke et al., 2016[Zinke, M., Hanff, E., Böhmer, A., Supuran, C. T. & Tsikas, D. (2016). Amino Acids, 48, 245-255.]). 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[Tsikas, D., Sandmann, J. & Frölich, J. C. (2002). J. Chromatogr. B, 772, 335-346.]).

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[Tu, C., Wynns, G. C. & Silverman, D. N. (1981). J. Biol. Chem. 256, 9466-9470.]). 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[Hanff, E., Böhmer, A., Zinke, M., Gambaryan, S., Schwarz, A., Supuran, C.T. & Tsikas, D. (2016). Amino Acids, 48, 1695-706.]; Zinke et al., 2016[Zinke, M., Hanff, E., Böhmer, A., Supuran, C. T. & Tsikas, D. (2016). Amino Acids, 48, 245-255.]).

Andring et al. (2020[Andring, J. T., Kim, C. U. & McKenna, R. (2020). IUCrJ, 7, 287-293.]) stated that `recent reports have shown that CAII can also reduce nitrite (NO2) to nitric oxide (NO)... (Andring et al., 2018[Andring, J. T., Lomelino, C. L., Tu, C., Silverman, D. N., McKenna, R. & Swenson, E. R. (2018). Free Radical Biol. Med. 117, 1-5.]; Aamand et al., 2009[Aamand, R., Dalsgaard, T., Jensen, F. B., Simonsen, U., Roepstorff, A. & Fago, A. (2009). Am. J. Physiol. Heart Circ. Physiol. 297, H2068-H2074.]; Hanff et al., 2018[Hanff, E., Zinke, M., Böhmer, A., Niebuhr, J., Maassen, M., Endeward, V., Maassen, N., & Tsikas, D. (2018). Anal. Biochem. 550, 132-136.])', 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[Hanff, E., Zinke, M., Böhmer, A., Niebuhr, J., Maassen, M., Endeward, V., Maassen, N., & Tsikas, D. (2018). Anal. Biochem. 550, 132-136.])', 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[Andring, J. T., Lomelino, C. L., Tu, C., Silverman, D. N., McKenna, R. & Swenson, E. R. (2018). Free Radical Biol. Med. 117, 1-5.]; Hanff et al., 2018[Hanff, E., Zinke, M., Böhmer, A., Niebuhr, J., Maassen, M., Endeward, V., Maassen, N., & Tsikas, D. (2018). Anal. Biochem. 550, 132-136.]).'. We wish to point out this mistake in the paper by Andring et al. (2020[Andring, J. T., Kim, C. U. & McKenna, R. (2020). IUCrJ, 7, 287-293.]). In the paper referred to above (i.e., Hanff et al., 2018[Hanff, E., Zinke, M., Böhmer, A., Niebuhr, J., Maassen, M., Endeward, V., Maassen, N., & Tsikas, D. (2018). Anal. Biochem. 550, 132-136.]), 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[Hanff, E., Zinke, M., Böhmer, A., Niebuhr, J., Maassen, M., Endeward, V., Maassen, N., & Tsikas, D. (2018). Anal. Biochem. 550, 132-136.]).

References

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IUCrJ
Volume 8| Part 2| March 2021| Pages 327-328
ISSN: 2052-2525