Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase: ultrafast spectroscopy of a Trp-->Phe mutant.
Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH* in DNA photolyase from Escherichia coli into FADH- involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADH*-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH* was found to decay in approximately 30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH* fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382*+ and (in WT) electron transfer from W359 to W382*+ occur with time constants <4 ps, considerably faster than the initial W382-->FADH* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of approximately 10(-12) s.
Lukacs
Andras
A
Eker
André P M
AP
Byrdin
Martin
M
Villette
Sandrine
S
Pan
Jie
J
Brettel
Klaus
K
Vos
Marten H
MH
D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000302:isolation & purification; D004579:Electron Transport; D004926:Escherichia coli / Q000737:chemistry; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014364:Tryptophan / Q000737:chemistry*
D005609:Free Radicals; D005182:Flavin-Adenine Dinucleotide; D010649:Phenylalanine; D014364:Tryptophan; D004255:Deoxyribodipyrimidine Photo-Lyase