title: Theoretical Studies of the Electronic Absorption Spectra of Thiamin Diphosphate in Pyruvate Decarboxylase.
abstract: Electronic absorption spectra are oftentimes used to identify reaction intermediates or substrates/products in enzymatic systems, as long as absorption bands can be unequivocally assigned to the species being studied. The latter task is far from trivial given the transient nature of some states and the complexity of the surrounding environment around the active site. To identify unique spectral fingerprints, controlled experiments with model compounds have been used in the past, but even these can sometimes be unreliable. Circular dichroism (CD) and ultraviolet-visible spectra have been tools of choice in the study of the rich chemistry of thiamin diphosphate-dependent enzymes. In this study, we focus on the Zymomonas mobilis pyruvate decarboxylase, and mutant analogues thereof, as a prototypical representative of the thiamin diphosphate (ThDP) enzyme superfamily. Through the use of electronic structure methods, we analyze the nature of electronic excitations in the cofactor. We find that all the determining CD bands around the 280-340 nm spectral range correspond to charge-transfer excitations between the pyrimidine and thiazolium rings of ThDP, which, most likely, is a general property of related ThDP-dependent enzymes. While we can confirm the assignments of previously proposed bands to chemical states, our calculations further suggest that a hitherto unassigned band of enzyme-bound ThDP reports on the ionization state of the canonical glutamate that is required for cofactor activation. This finding expands the spectroscopic "library" of chemical states of ThDP enzymes, permitting a simultaneous assignment of both the cofactor ThDP and the activating glutamate. We anticipate this finding to be helpful for mechanistic analyses of related ThDP enzymes.
authors.affiliation: Institute of Physical Chemistry, University of Goettingen , Tammannstraße 6, D-37077 Göttingen, Germany.
authors.forename: Mirko
authors.initials: M
authors.lastname: Paulikat
authors.affiliation: Department of Molecular Enzymology, Albrecht-von-Haller-Institute for Plant Sciences, and Göttingen Center for Molecular Biosciences, University of Goettingen , Julia-Lermontowa-Weg 3, D-37077 Göttingen, Germany.
authors.forename: Cindy
authors.initials: C
authors.lastname: Wechsler
authors.affiliation: Department of Molecular Enzymology, Albrecht-von-Haller-Institute for Plant Sciences, and Göttingen Center for Molecular Biosciences, University of Goettingen , Julia-Lermontowa-Weg 3, D-37077 Göttingen, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Tittmann
authors.affiliation: Institute of Physical Chemistry, University of Goettingen , Tammannstraße 6, D-37077 Göttingen, Germany.
authors.forename: Ricardo A
authors.identifier: 0000-0002-2720-3364
authors.initials: RA
authors.lastname: Mata
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017433:Protein Structure, Secondary; D011743:Pyrimidines / Q000737:chemistry; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism; D017355:Zymomonas / Q000737:chemistry* / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D011743:Pyrimidines; D018698:Glutamic Acid; D011767:Pyruvate Decarboxylase; C030986:pyrimidine; D013835:Thiamine Pyrophosphate