Pulse radiolysis studies on galactose oxidase

Single-Cu-containing galactose oxidase in the GOase(semi) state (Cu(II), no Tyr(*) radical) reacts with pulse radiolysis generated formate radicals CO(2)(*-) to give an intermediate UV-vis spectrum assigned as RSSR(*-), peak at 450 nm (epsilon = 8100 M(-1) cm(-1)). From a detailed kinetic analysis at 450 nm, pH 7.0, the following steps have been identified. First the strongly reducing CO(2)(*-) (-1.9V) reduces GOase(semi) (k(0) > or = 6.5 x 10(8) M(-1) s(-1)) to a species GOase(semi)(*-). This is followed by biphasic reactions (i) GOase(semi)(*-) + GOase(semi) (k(1) = 1.6 x 10(7) M(-1) s(-1)) to give GOase(semi) + P(*-) and (ii) P(*-) + GOase(semi) (k(2) = 6.7 x 10(6) M(-1) s(-1)) to give GOase(semi)RSSR(*-). There are no significant absorbance changes for the formation of GOase(semi)(*-) and P(*-), which are Cu(I) (or related) species. However, GOase(semi)RSSR(*-) has an absorption spectrum which differs significantly from that of GOase(semi). The 450 nm peak is characteristic of an RSSR(*-) radical with two cysteines in close sequence proximity and is here assigned to Cys515-Cys518, which is at the GOase surface and 10.2 A from the Cu. On chemical modification of the RSSR group with HSPO(3)(2-) to give RSSPO(3)H(-) and RS(-), absorbance changes are approximately 50% of those previously observed. The decay of RSSR(*-) (0.17 s(-1)) results in the formation of GOase(red). No RSSR(*-) formation is observed in the reaction of GOase(semi) Tyr495Phe with CO(2)(*-), and a single process giving GOase(red)Tyr495Phe occurs. Similarly in the reaction of GOase(ox) with CO(2)(*-), a single-stage reaction gives GOase(semi).