Flavin methyl protons

Another interesting feature of the flavin enhancements visible at short delays in Figure 17, is the appearance of a line (F,) from the methyl protons at position 7 of the flavin ring. This peak had never been observed in the argon laser spectra even under conditions of pH and concentration where the flavin signals were rather strong. The very small... 

The back reaction (a) could in principle explain the observed results but for the initial growth of Ijj(t) expected from F-pair polarization. In addition this mechanism fails to explain the small time dependence of the tryptophan polarization. It is reasonable to expect that Tj for a ring proton in tryptophan would be somewhat smaller than for a ring-methyl proton in flavin simply because of the r dependence of the electron-proton dipolar interaction. Therefore as shown in Figure 7 for short Tj, a predominantly increasing Ijj(t) would be predicted for tryptophan in contrast to the observed results. 

LSDl, also known as BHCllO, is the first lysine specific demethylase that was discovered. It has been assigned to group I of lysine demethylases (KDMl) [90, 91]. LSDl contains an amine oxidase domain responsible of the enzymatic activity and has been isolated as a stable component from several histone modifying complexes. The enzymatic characterization of this protein revealed that FAD (flavine adenine dinucleotide) is required as a cofactor for the removal of the methyl group. Furthermore, LSDl requires a protonated nitrogen in order to initiate demethylation so that this enzyme is only able to demethylate mono- or dimethylated substrates but not trimethylated substrates [98, 99]. 

G [139], These widths correlate with the optical spectra of the radicals semiquin-ones with a 19 G linewidth have pronounced absorption between 550 and 650 nm and appear blue those with a 15 G linewidth (Fig. 8a) have spectra with peaks between 485 and 370 nm and appear red. The additional width of the 19 G spectrum is due to an exchangeable proton it has been shown that the linewidth decreases to 15 G in DjO solutions. Experiments on model compounds [141] indicate that the blue type of radical is a neutral semiquinone with the proton on N(5) of the isoalloxazine ring (9), and that the red species is either the semiquinone anion or the neutral 0(4)-enol tautomer. Covalently bound semiquinones have ESR spectra that are distinctly narrowed [142-144], having a width of around 12 G (Fig. 4b). The reason for this is that the covalently bound flavins lack a methyl group at C-8, which when present makes a significant contribution to the total linewidth. 

Over the years there have been a number of mechanistic proposals for substrate oxidation by TMADH. An early proposal considered a carbanion mechanism in which an active site base deprotonates a substrate methyl group to form a substrate carbanion [69] reduction of the flavin was then achieved by the formation of a carbanion-flavin N5 adduct, with subsequent formation of the product imine and dihydroflavin. A number of active site residues were identified as potential bases in such a reaction mechanism. Directed mutagenesis and stopped-flow kinetic studies, however, have been used to systematically eliminate the participation of these residues in a carbanion-type mechanism [76-79], thus indicating that a proton abstraction mechanism initiated by an active site residue does not occur in TMADH. Early proposals also invoked the trimethylammonium cation as the reactive species in the enzyme-substrate complex, owing to the high (9.81) of free... 

Figure 11 shows the result of this experiment on a solution of 5 mM N-acetyl tryptophan and 0.2 mM 3-N-carboxy-methyl lumiflavin, hereafter simply called flavin (see Figure 10). Positive enhancements can be observed for the aromatic C-2, C-4 and C-6 protons, while the CH2 group shows emission. This polarization pattern corresponds with a tryptophyl radical in which the electron spin is delocalized over the aromatic ring. It can further be noted that almost no flavin polarization is present in the difference spectrum. Figure 11c (weak lines are present at 2.6 and 4.0 ppm). This is due to cancellation of recombination and escape polarization as will be discussed in Section 5. The mechanism of the photoreaction undoubtedly involves triplet flavin (17). Since 1-N-methyl tryptophan shows similar CIDNP effects, the primary step most probably is electron transfer to the photo-excited flavin. This is also supported by a flash photolysis study by Heelis and Phillips (18). The nature of the primary step in the photoreactions with amino acids is important in view of the interpretation of "accessibility" of an amino acid side chain in a protein as seen by the photo-CIDNP method. This question is therefore the subject of further study.

In contrast to the case of tryptophan the photoreactions with tyrosine and histidine probably involve hydrogen atom transfer as the primary step. There are several indications for this. First, 0-methylated tyrosine (p-methoxy phenylalanine) did not show any photo-CIDNP effect and its reactivity as a photo-reductant towards flavins is strongly reduced (19). Similarly, 1-N-methyl histidine is not polarized at high pH (> 7.5), when no abstractable hydrogen is present. Secondly, in the protein ribonuclease A, which has a well known 3-dimensional structure, the residues Tyr 92 and His 105 have exposed rings, but their OH and NH protons are hydrogen bonded to backbone carbonyl groups. 

The a-CH polarization is still another exajqple of polarization transfer by cross-relaxation (In this case from the B CH2 group). The splitting of the B-CH2 protons by 0.2 ppm corresponds with a conformational change upon NAG binding, whereby one proton moves towards the plane of Trp 63 1 approximately 0.1 8, whereas the other proton moves away from this plane over the same distance. Smaller shifts occur for the lines of the other tryptophan. Interestingly the flavin 10-methyl line at 4.0 ppm... 

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