Resonance Raman spectroscopy of ribonucleotide reductase. Evidence for a deprotonated tyrosyl radical and photochemistry of the binuclear iron center.
Native ribonucleotide reductase from Escherichia coli exhibits a resonance-enhanced Raman mode at 1498 cm-1 that is characteristic of a tyrosyl radical. The Raman frequency as well as the absorption maximum at 410 nm identifies the radical as being in a deprotonated state. The B2 subunit of ribonucleotide reductase shows an additional resonance Raman mode at 493 cm-1 that has been assigned to the symmetric stretch of an Fe-O-Fe moiety. When samples of active B2 or metB2 are exposed to a tightly focused laser beam at 406.7 nm, there is a loss of intensity at 493 cm-1 and the appearance of a new peak at 595 cm-1. Although the 595-cm-1 feature was previously assigned to an Fe-OH vibration on the basis of its 23-cm-1 shift to lower energy in H2(18)O and the apparent dependence of its intensity on pH [Sjöberg, B. M., Loehr, T. M., & Sanders-Loehr, J. (1987) Biochemistry 26, 4242], the present studies indicate that the intensity of this mode is dependent primarily on input laser power. The peak at 595 cm-1 is more plausibly assigned to a new vs(Fe-O-Fe) mode in view of its lack of the deuterium isotope dependence expected for an Fe-OH mode and its resonant scattering cross section which is comparable to that of the 493-cm-1 mode. This new species has a calculated Fe-O-Fe angle of approximately 113 degrees compared to approximately 138 degrees calculated for the Fe-O-Fe unit in unmodified protein B2. One possible explanation for the photoinduced vibrational mode is that a bridging solvent molecule has been inserted in place of a bridging carboxylate.
Backes
G
G
Department of Chemical and Biological Sciences, Oregon Graduate Center, Beaverton 97006-1999.
Sahlin
M
M
Sjöberg
B M
BM
Loehr
T M
TM
Sanders-Loehr
J
J
D004926:Escherichia coli / Q000201:enzymology; D005609:Free Radicals; D007501:Iron / Q000378:metabolism; D011487:Protein Conformation; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D012997:Solvents; D013059:Spectrum Analysis, Raman / Q000379:methods; D014443:Tyrosine
D005609:Free Radicals; D012997:Solvents; D014443:Tyrosine; D007501:Iron; D012264:Ribonucleotide Reductases