Probing the N(5)-H bond of the isoalloxazine moiety of flavin radicals by X- and W-band pulsed electron-nuclear double resonance.
An X- (9.7 GHz and W-band (94 GHz) pulsed electron-nuclear double resonance (ENDOR) study of the flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form is presented. Through proton and deuteron ENDOR measurements at T = 80 K, we detect and characterize the full anisotropy of the hyperfine coupling (hfc) tensor of the proton or deuteron bound to N(5) of the isoalloxazine ring. Scaling of the anisotropic proton hfc components by multiplication with the quotient of the magnetogyric ratio of a deuteron and a proton, chiD/chiH, reveals subtle differences compared to the respective deuteron couplings obtained by 95-GHz deuterium ENDOR spectroscopy on an H-->D buffer-exchanged sample. These differences can be attributed to the different lengths of N(5)-H and N(5)-D bonds arising from the different masses of protons and deuterons. From the R(-3) dependence of the dipolar hyperfine splitting, we estimated that the N(5)-D bond is about 2.5% shorter than the respective N(5)-H bond. That such subtle bond-length differences can be resolved by pulsed ENDOR spectroscopy suggests that this method may be favorably used to probe the geometry of hydrogen bonds between the H(5) of the paramagnetic flavin and the protein backbone. Such information is only obtained with difficulty by other types of spectroscopy.
Weber
Stefan
S
Freie Universität Berlin, Fachbereich Physik, Arnimallee 14, 14195 Berlin, Germany. stefan.weber@physik.fu-berlin.de
Kay
Christopher W M
CW
Bacher
Adelbert
A
Richter
Gerald
G
Bittl
Robert
R
D016880:Anisotropy; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry; D003903:Deuterium / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000295:instrumentation / Q000379:methods*; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005415:Flavins / Q000737:chemistry*; D006859:Hydrogen / Q000737:chemistry*; D006860:Hydrogen Bonding; D008956:Models, Chemical; D015394:Molecular Structure; D009584:Nitrogen / Q000737:chemistry*; D016011:Normal Distribution; D011522:Protons; D013052:Spectrometry, X-Ray Emission; D013053:Spectrophotometry; D013696:Temperature
D005415:Flavins; D011522:Protons; C008173:isoalloxazine; D006859:Hydrogen; D003903:Deuterium; D004255:Deoxyribodipyrimidine Photo-Lyase; D009584:Nitrogen