Energy transduction during catalysis by Escherichia coli DNA photolyase.
Native DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. Quantum yield and action spectral data for thymine dimer repair were obtained by using a novel multiple turnover approach under aerobic conditions. This method assumes that catalysis proceeds via a (rapid-equilibrium) ordered mechanism with light as the second substrate, as verified in steady state kinetic studies. The action spectrum observed with native enzyme matched its absorption spectrum and an action spectrum simulated based on an energy transfer mechanism where dimer repair is initiated either by direct excitation of FADH2 or by pterin excitation followed by singlet-singlet energy transfer to FADH2. The quantum yield observed for dimer repair with native enzyme (phi Native = 0.722 +/- 0.0414) is similar to that observed with enzyme containing only FADH2 (phi EFADH2 = 0.655 +/- 0.0256), as expected owing to the high efficiency of energy transfer from the natural pterin to FADH2 [EET = 0.92]. The quantum yield observed for dimer repair decreased (2.1-fold) when the natural pterin was partially (68.8%) replaced with 5,10-CH(+)-H4folate (phi obs = 0.342 +/- 0.0149). This is consistent with the energy transfer mechanism (phi calc = 0.411 +/- 0.0118) since a 2-fold lower energy transfer efficiency is observed when the natural pterin is replaced with 5,10-CH(+)-H4folate (EET = 0.46) (Lipman & Jorns, 1992). The action spectrum observed for 5,10-CH(+)-H4folate-supplemented enzyme matched a simulated action spectrum which exhibited a small (5 nm) hypsochromic shift as compared with the absorption spectrum (lambda max = 385 nm).(ABSTRACT TRUNCATED AT 250 WORDS)
Ramsey
A J
AJ
Department of Biological Chemistry, Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102.
Alderfer
J L
JL
Jorns
M S
MS
D002384:Catalysis; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics
D005182:Flavin-Adenine Dinucleotide; C058805:1,5-dihydro-FAD; D004255:Deoxyribodipyrimidine Photo-Lyase