A “How-To” Guide to the Stark Spectroscopy of Flavins and Flavoproteins
Flavins and flavoproteins have been studied by a plethora of spectroscopic techniques. Beginning with the characterization of DNA photolyases and the discovery of the diversity of roles played by excited-state flavins in photobiology, the characterization of the electronic excited state of flavins has become increasingly important. In this protocol, we provide a guide to using Stark spectroscopy in obtaining the degree of electronic charge redistribution in simple flavins and in flavoproteins. Stark spectroscopy is technically simpler than more common approaches used to explore the structure of the excited state, considerably cheaper to implement, and yet very powerful in its scope. At the end of this guide, we present data taken on non-photobiological flavoproteins, glutathione reductase and lipoamide dehydrogenase, that suggest that Stark spectroscopy is a unique way to elucidate the electrostatic environment that the flavin cofactor experiences bound inside the protein.
Key wordsStark spectroscopy Excited-state electronic structure Flavins Flavoproteins Electroabsorption Low-temperature absorption spectroscopy
Dr. Goutham Kodali gave very useful feedback on this manuscript. We wish to thank Dr. Ron Koder for the TPARF and Dr. Nancy Hopkins for the lipoamide dehydrogenase enzyme. R.P. and R.J.S. were supported by a grant from the NSF Division of Chemistry (CHE-0847855). R.J.S. is grateful for support from the NSF Molecular Biosciences Division (MCB-0347087).
- 1.Kay CWM, Bacher A, Fischer M, Richter G, Schleicher E, Weber S (2006) Blue light-initiated DNA repair by photolyase. Compr Series Photochem Photobiol Sci 6:151–182Google Scholar
- 4.Kennis JTM, Alexandre MTA (2006) Mechanisms of light activation in flavin-binding photoreceptors. Compr Series Photochem Photobiol Sci 6:287–319Google Scholar
- 6.Matsika S (2007) Conical intersections in molecular systems. Rev Comput Chem 23:83–124Google Scholar
- 9.Sokolova O, Cecala C, Gopal A, Cortazar F, McDowell-Buchanan C, Sancar A, Gindt YM, Schelvis JPM (2007) Resonance Raman spectroscopic investigation of the light-harvesting chromophore in Escherichia coli photolyase and Vibrio cholerae cryptochrome-1. Biochemistry 46:3673–3681PubMedCrossRefGoogle Scholar
- 20.Liptay W (1974) Dipole moments and polarizabilities of molecules in excited electronic states. In: Lim EC (ed) Excited states. Academic, New York, pp 129–229Google Scholar
- 39.Cai ZL, Sevilla MD (2004) Studies of excess electron and hole transfer in DNA at low temperatures. Topics in Current Chemistry, Long-range charge transfer in DNA II 237:103–127Google Scholar
- 45.Press WH, Flannery BP, Teukolsky SA, Vetterling WT (1988) Numerical recipes in C: the art of scientific computing. Cambridge University Press, New YorkGoogle Scholar
- 46.Böttcher CJF (1952) Theory of electric polarization. Elsevier, HoustonGoogle Scholar
- 53.Krauth-Siegel RL, Lohrer H, Hungerer KD, Schoellhammer T (1991) Lipoamide dehydrogenase and trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas’ disease. In: Flavins Flavoproteins Proc. Int. Symp., 10th, pp 843–846Google Scholar
- 54.Williams CH Jr (1992) Lipoamide dehydrogenase, glutathione reductase, thioredoxin reductase, and mercuric ion reductase. A family of flavoenzyme transhydrogenases. Chem Biochem Flavoenzymes 3:121–211Google Scholar
- 56.Kodali G (2009) Excited state electronic properties of DNA photolyase and fluorescent nucleobase analogues (FBA): An experimental and theoretical study. UMI Dissertation Publishing 1–266Google Scholar