Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase

The Escherichia coli class Ia ribonucleotide reductase (RNR) achieves forward and reverse proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (beta-Y-122 (sic) beta-Y-356 (sic) alpha-Y-730 (sic) alpha-C-439) spanning similar to 35 angstrom and two subunits every time it turns over. We have developed photoRNRs that allow radical transport to be photo-triggered at tyrosine (Y) or fluorotyrosine (FnY) residues along the PCET pathway. We now report a new photoRNR in which photooxidation of a tryptophan (W) residue replacing Y-356 within the alpha/beta subunit interface proceeds by a stepwise ET/PT (electron transfer then proton transfer) mechanism and provides an orthogonal spectroscopic handle with respect to radical pathway residues Y-731 and Y-730 in alpha. This construct displays an similar to 3-fold enhancement in photochemical yield of W-center dot relative to F3Y center dot and a similar to 7-fold enhancement relative to Y-center dot. Photogeneration of the W-center dot radical occurs with a rate constant of (4.4 +/- 0.2) x 10(5) s(-1), which obeys a Marcus correlation for radical generation at the RNR subunit interface. Despite the fact that the Y --> W variant displays no enzymatic activity in the absence of light, photogeneration of W-center dot within the subunit interface results in 20% activity for turnover relative to wild-type RNR under the same conditions.