Research article

Reduced Flavin: NMR investigation of N(5)-H exchange mechanism, estimation of ionisation constants and assessment of properties as biological catalyst

Peter Macheroux¹ , Sandro Ghisla² , Christoph Sanner³ , Heinz Rüterjans³ and Franz Müller⁴

¹Graz University of Technology, Institute of Biochemistry, Petersgasse 12, A-8010 Graz, Austria

²Fachbereich Biologie, Universität Konstanz, D-78457 Konstanz, Germany

³Institut fur Biophysikalische Chemie, J.W. Goethe-Universität, Biozentrum N230, Marie-Curie-Strasse 9, D-60439 Frankfurt am Main, Germany

⁴Wylstrasse 13, CH-6052 Hergiswil, Switzerland

author email corresponding author email

BMC Biochemistry 2005, 6:26doi:10.1186/1471-2091-6-26

Published:	25 November 2005

Abstract

Background

The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context.

Results

N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the ¹⁵N-NMR-signals of ¹⁵N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the ¹⁵N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the ¹⁵N(5) signal and the proton exchange rates are little dependent on the buffer system used.

Conclusion

The exchange rates allow an estimation of the pK_a value of N(5)-H deprotonation in reduced flavin to be ≥ 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK ≈ 4 for N(5)-H protonation (to form N(5)⁺-H₂) would be consistent with a role of N(5)-H as a base.