On the electronic structure of free and protein-bound isoalloxazines

The subject of this thesis, as apparent from its title, is a detailed study of the isoalloxazine molecule by a variety of techniques belonging to the field of optical spectroscopy. After a brief introduction to the subject in general terms, together with some historical background and citation of relevant review papers which appeared before 1979 (Chapter 1), a general outline of experimental techniques and theoretical methods used is given (Chapter 2). More detailed information about specific experiments or calculations is provided in the experimental sections of the individual chapters.Chapters 3 and 4 describe the continuous wave and time-resolved spectral properties of (iso)alloxazines determined under experimental conditions, in which external perturbation of the molecules of interest is minimized as much as possible. This could be achieved by measurements in apolar solvents (Chapter 3) and in the vapour phase (Chapter 4), a state of aggregation in which the molecule could be brought with some experimental skill. Thus, a large temperature range (77 to 520 K) could be employed. In solution at 77 and 300 K (Chapter 3), the spectra revealed a 1250 cm ^-1vibrational progression in all isoalloxazines investigated (probably a stretching mode); actual lifetimes of 5 - 10 ns for fluorescence and ~ 300 ms for phosphorescence (77 K, no phosphorescence could be detected in fluid solution) were observed; the ratio of the actual and radiative lifetimes of the electronically first excited singlet state agreed very well with an independent quantum yield determination and solvent interactions were found to affect primarily the Franck-Condon envelopes of the spectra and not the electronic transition energies. The non-radiative decay of isoalloxazine, on the other hand, is strongly dependent on the molecule's environment. An anomalously large Stokes-loss in fluid and glassy solution is indicative of a conformational change of the molecule occurring upon electronic excitation. In alkane solution at 77 K, isoalloxazines form clusters exhibiting P-type (triplet-triplet annihilation process) delayed fluorescence. Vapour phase spectra (Chapter 4) , although less structured than solution spectra owing to sequence congestion, provided primary information on isolated (iso)alloxazines when comparison with spectra obtained in condensed media was made. Fluorescence lifetimes range from < 0.5 ns for alloxazine to 1-18 ns for isoalloxazine vapour. The data indicate possible intramolecular complex formation between the isoalloxazine ring system and its own aliphatic side-chain carrying a (polar) hydroxyl group. Direct photodissociation of isoalloxazine in the electronically first excited singlet state is not a probable process in the vapour phase.Chapter 5 describes the continuation of (iso)alloxazine vapour phase experiments by the application of ultraviolet photoelectron spectroscopy. Both He(I) and He(II) excitation was used. The spectra are interpreted using various methyl substituted isoalloxazines and by comparison with the results obtained from CNDO/S calculations and photoionization cross- sections derived therefrom. The dependence of the electronic properties of isoalloxazines on the redox state and the degree of substitution is analyzed. A critical review of the data obtained from semiempirical MO calculations by various other authors and the CNDO/S results shows that a few methods give a fairly good prediction of the πorbital energies only. Without exception, the calculated σorbital energies contain considerable error. Particularly, all theoretical methods fail to predict a threefold degeneracy in the orbital level scheme at ~-9.6 eV, in which both πand σorbitals are involved. Possible reasons for the failures are discussed. Analysis of the experimental and theoretical results reveals a planar molecular conformation to be the most probable me for an isolated reduced isoalloxazine molecule in the vapour phase, contrary to the bent conformation which is encountered in solution or the solid state. Bending is, therefore, most likely caused by interaction of the molecule with its environment. Such interactions, leading to changes in orbital energies, may in part be responsible for the ability of the protein-bound flavocoenzyme to be involved in a broad diversity of biological reactions.In chapter 6, all foregoing data are put together in a combined analysis to establish relationships between the photoelectron and ordinary optical spectra. First, additional photoelectron spectra of 10-hydroxyalkyl-isoalloxazines confirmed the existence of the intramolecular complex proposed on the basis of vapour phase fluorescence lifetimes (vide supra). The hydrogen-bond between the side-chain OH group and the N ₁ imine-like nitrogen atom (cf. Scheme 1.1, p. 1) leads to a destabilization of the lone pair on the latter atom, contrary to common experience. This anomalous behaviour can be ascribed to rearrangement of existing through- bond interactions in the free isoalloxazine molecule. Results from CNDO/S and INDO/S calculations agree reasonably with the observations. Secondly these findings have considerable impact on the interpretation of existing and in this thesis presented optical spectra. The new photoelectron spectroscopic data lead, in combination with optical spectra, to the assignment of the isoalloxazine S ₂ to a mixed state containing (anomalous) ny* character arising from the anomalous behaviour of the N ₁ lone pair (cf. Scheme 1.1, p.1). The oscillator strength of the
S ₀ ->S ₂  π-> π* component is very sensitive to external perturbation. Finally, the possible implications for biochemical catalysis by flavins are discussed.Chapter 7 describes a thorough investigation of Old Yellow Enzyme (OYE), a (flavo)protein containing the isoalloxazine derivative flavin mononucleotide (M). It is attempted to interpret the spectral properties of this protein in terms of intermolecular interactions between its constituents, using the knowledge and experience acquired in the preceeding research. The results demonstrate the inadequacy of the existing explanation of the phenomena occurring upon the addition of phenols to OYE , based on the formation of a simple phenolate -FMN donor- acceptor charge transfer complex. Instead,it was found that the phenolate anion interferes strongly with an existing tight complex between FMN and the apoprotein, probably a H-bonded structure in which FMN is tautomerized and interacts with a L-chiral center. This is concluded from a separate electronic transition with an origin at 496 m, thus far not recognized as such, and the circular dichroism observed. The emission of OYE is dominated by that of free FMN, although protein-bound FMN seems also to become luminescent in glassy solution at 143 K. A second fluorescence/phosphorescence emission appears upon UV-excitation of both native and complexed OYE . This emission is quenched by the addition of phenol to OYE , shows a large (3000 cm ^-1) blue shift on going to a low temperature glass and is tentatively assigned to excimers of nucleic acids. Long-wavelength excitation with a synchronously pumped, mode-locked Rhodamine 6G dye laser revealed a third, extremely weak, emission in both native OYE and its complexes. It decays with ~3 ns lifetime at 143 K. ESR spectra revealed the presence of a low amount of an unpaired spin in OYE. Owing to an unusual relaxational behaviour it could only be observed below 15 K and the signal was measured in both the free enzyme and its complexes. Possible assignment and consequences of this observation are discussed. In frozen aqueous solutions of the OYE-phenolate complex, a phase transition was discovered at which the colour reverted to that of the native enzyme. Subsequent melting restored the original colour. The observed phenomena and existing literature data lead to the conclusion that the only model from which no apparent inconsistencies emerge, is that of a very complicated network of hydrogen-bonded structures in the protein. These involve several, partly unknown, chromophores. Phenols interfere with this network, leading to the formation of the long- wavelength absorption band in OYE.Finally, a brief postscript (Chapter 8) is given, containing an overview of recent spectroscopic literature on isoalloxazines, a discussion in response to a polemic in papers by other scientists and the ESCA (XPS) spectrum of 3,7,8,10-tetramethyl-isoalloxazine (3-methyllumiflavin).