PULSE-RADIOLYSIS STUDIES OF INTRAMOLECULAR ELECTRON-TRANSFER IN MODELPEPTIDES AND PROTEINS .7. TRP-]TYRO RADICAL TRANSFORMATION IN HEN EGG-WHITE LYSOZYME - EFFECTS OF PH, TEMPERATURE, TRP62 OXIDATION AND INHIBITOR BINDING

Intramolecular long-range electron transfer (LRET) in hen egg-white ly sozyme (HEWL) accompanying Trp --> TyrO radical transformation was inv estigated in aqueous solution by pulse radiolysis as a function of pH (5.2-7.4) and temperature (283-328K). The reaction was induced by high ly selective oxidation of Trp with N-3(.) radicals under low concentra tion of the reactants but at a high HEWL/N-3(.) molar ratio, so that m ore than 99% of the oxidized protein molecules contained only a single tryptophyl radical. Synchronous decay of Trp(.) and build-up of TyrO( .) conformed satisfactorily to first-order kinetics, indicating that L RET involved either one or more Trp(.)/Tyr redox pairs characterized b y similar rate constants. The rate constant of LRET, k(5), increased m onotonously with decreasing pH showing the following characteristics: (i) in the pH range 7.4-5.2 the plot of k(5) against pH was sigmoidal in shape, reflecting protonation of Glu35 (pK(a) approximate to 6) and pointing to involvement of conformational control of the kinetics of LRET, (ii) below pH5.2 a sharp increase in k(5) was observed due to th e protonation of Trp(.) to form TrpH(.+), which is known to oxidize ty rosine faster than does Trp(.). Arrhenius plots of the temperature-dep endence of k(5) showed that the activation energy of LRET varies both with temperature and the protonation state of the enzyme. The activati on energies are in the range 7.6-56.0 kJ mol(-1) and are similar to th ose for activation of amide hydrogen exchange in native HEWL below its denaturation temperature. Selective oxidation by ozone of the Trp62 i ndole side-chain in HEWL to N'-formylkynurenine (NFKyn62-HEWL) caused a large drop in the initial yield of Trp(.) radicals, G(Trp(.))(i). Th is was accompanied by a relatively small decrease in k(5) but selectiv e oxidation by ozone had a pronounced effect on its temperature-depend ence. Taken together these observations indicate that of the six trypt ophans present in HEWL Trp62 contributes about 50% to the yield of the observed LRET. In the enzyme-inhibitor complex, HEWL(GlcNAc)(3), wher e Trp62 and Trp63 are completely shielded from the solvent by the boun d triacetylchitotriose, G(Trp(.))(i) was lower than in NFKyn62-HEWL, a nd both the kinetic and energetic characteristics of LRET, observed at pH5.2, were again somewhat different than in HEWL alone. Considering known solvent accessibilities of tryptophans in the complex, the obser ved LRET process in HEWL(GlcNAc)(3) was assigned to Trp123. Theoretica l evaluation of the electronic coupling for the dominant LRET pathways between all the potential Trp(.)/Tyr redox couples in HEWL, with help of the PATHWAYS model, enabled Trp62/Tyr53, Trp63/Tyr53 and Trp123/Ty r23 to be identified as the pairs involved in the experimentally obser ved electron transfer.