Looking at other GRE for which H/D exchange experiments have been reported, the samples of purified and in-vitro-activated PFL contained 2 mM substrate or substrate analog (pyruvate or oxamate) as a necessary component of the activation reaction . Moreover, the covalently bound pyruvate in PFL is attached at Cys418, enabling the presence of bound substrate during the Cys419-mediated H/D exchange at the glycyl radical C atom . In contrast, ARNR was essentially assayed in the apoenzyme state, i.e. with only formate added as a cosubstrate for the activation reaction, but without nucleotide triphosphate, which was correlated with the absence of observable H/D exchange .