A NEW APPROACH TO THE STUDY OF INTRAMOLECULAR ELECTRON-TRANSFER REACTIONS OF METALLOPROTEINS - PULSE-RADIOLYSIS OF NO2-MODIFIED TYROSINE DERIVATIVES OF PLASTOCYANIN

Single NO2-modified derivatives of plastocyanin, with modifications at Tyr83 (spinach) and Tyr83/Tyr62 (parsley, deletions at 57 and 58), have been prepared and characterized by peptide mapping. All three products give a TyrNO2 UV-vis absorbance band at 355 nm (epsilon = 3900 M-1 cm-1) at pH <7.5, assigned to NO2, which shifts to 428 nm on increasing the pH to >9.0 due to acid dissociation of the phenolic group. Pulse radiolysis experiments in which the reductant CO2.- (or Cd+) is generated give with all three PCuIITyrNO2 derivatives one observable reaction step in which PCuII is reduced to PCuI. Rate constants are of order of magnitude 10(8) M-1 s-1 (CO2.-) and 10(9) M-1 s-I (Cd+). No PCuIITyrNO2.- radical intermediate absorbing at approximately 300 nm (epsilon approximately 10(4) M-1 cm-1) is observed. However in studies with PCuITyrNO2, reduction of the NO2 group is the only reaction step, with rate constants of the order of magnitude 10(9) M-1 s-1 (Cd+), and PCuITyrNO2.- is a relatively long-lived product. There is no crystallographic or spectroscopic evidence to suggest any Cu interaction with the phenolate or vice versa (separation >8.6 angstrom). If this is the case, it can be assumed that TyrNO2.- is generated at about the same rate in experiments on PCuIITyrNO2 and that rapid > 10(7) s-1 intramolecular electron transfer PCuIITyrNO2.- --> PCuITyrNO2 folloWS. We note that the rate constant for formation of PCuITyrNO2.- is of the same magnitude as the difference in rate constants for CO2. (or Cd+) with native PCUII and PCuIITyrNO2. Also the driving force for PCuIITyrNO2.- --> PCuITyrNO2 of approximately 800 mV (0.8 eV) favors rapid intramolecular electron transfer. The Beratan-Onuchic pathway program has been used to examine the best electron-transfer pathways from the Tyr83 and Tyr62 derivatives, and favorable through-bond pathways have been identified.