Single-Cu-containing galactose oxidase in the GOase(semi) state (Cu-II, no Tyr(.) radical) reacts with pulse radiolysis generated formate radicals CO2.- 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 rim, pH 7.0, the following steps have been identified. First the strongly reducing CO2.- (-1.9V) reduces GOase(semi) (k(0) greater than or equal to 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 GOases(emi)(.-) and P.-, which are Cu-1 (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 HSPO32- to give (RSSPOH-)-H-3 and RS-, absorbance changes are similar to50% 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 CO2.-, and a single process giving GOase(red)Tyr495Phe occurs. Similarly in the reaction of GOase(0x), with CO2.-, a single-stage reaction gives GOase(semi).