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One Protein, Two Chromophores: Comparative Spectroscopic Characterization of 6,7-Dimethyl-8-ribityllumazine and Riboflavin Bound to Lumazine Protein
journal contribution
posted on 2014-11-20, 00:00 authored by Bernd Paulus, Boris Illarionov, Daniel Nohr, Guillaume Roellinger, Sylwia Kacprzak, Markus Fischer, Stefan Weber, Adelbert Bacher, Erik SchleicherWe investigated the lumazine protein
from Photobacterium
leiognathi in complex with its biologically active cofactor,
6,7-dimethyl-8-ribityllumazine, at different redox states and compared
the results with samples containing a riboflavin cofactor. Using anaerobic
photoreduction, we were able to record optical absorption kinetics
from both cofactors in similar protein environments. It could be demonstrated
that the protein is able to stabilize a neutral ribolumazine radical
with ∼35% yield. The ribolumazine radical state was further
investigated by W-band continuous-wave EPR and X-band pulsed ENDOR
spectroscopy. Here, both the principal values of the g-tensor and an almost complete mapping of the proton hyperfine couplings
(hfcs) could be obtained. Remarkably, the g-tensor’s
principal components are similar to those of the respective riboflavin-containing
protein; however, the proton hfcs show noticeable differences. Comparing
time-resolved optical absorption and fluorescence data from ribolumazine-containing
samples, solely fluorescence but no signs of any intermediate radical
or a triplet state could be identified. This is in contrast to lumazine
protein samples containing the riboflavin cofactor, for which a high
yield of the photogenerated triplet state and some excited flavin
radical could be detected using time-resolved spectroscopy. These
results clearly demonstrate that ribolumazine is a redox-active molecule
and could, in principle, be employed as a cofactor in other enzymatic
reactions.