Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Multiple cysteins have been postulated to play a key role in this process. To test the role of various cysteines in nucleotide reduction, a variety of single and double mutants of the R1 subunit were prepared: C754S, C759S, C754-759S, C462S, C462A, C230S, and C292S. Due to the expression system, each mutant contains small amounts of contaminating wt-R1 (estimated to be 1.5-3% based on activity). An epitope tagging method in conjunction with anion exchange chromatography was used to partially resolve the mutant R1 from the wt-R1. The interaction of these mutants with the normal substrate was studied, which allowed a model to be proposed in which five cysteines of the R1 subunit of RDPR play a role in catalysis. C754S and C759S R1s catalyze CDP formation at rates similar to wt-R1 when DTT is used as a reductant. However, when thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH is used as reductant, the rates of dNDP production are similar to those expected for contaminating wt-R1 present as a heterodimer with the mutant. The impaired nature of these mutants with respect to reduction by TR suggests that their function is to transfer reducing equivalents from TR to the active site disulfide of R1 produced during NDP reduction. Single-turnover experiments, designed to avoid the problem of contaminating wt-R1, also support this role for C754 and C759. The double serine mutant of 754 and 759 has catalytic activity with DTT that is one-third the rate of wt-R1 with thioredoxin. C225 and C462 are thought to be the active site cysteines oxidized concomitantly with NDP reduction. Conversion of these cysteines to serines results in R1 mutants which convert the normal substrate into a mechanism-based inhibitor. C462SR1 upon incubation with R2 and [3'-3H,U-14C]UDP results in uracil release, 3H2O production, 3H,14C-labeled protein which has an absorbance change at 320 nm, and slow loss of the tyrosyl radical on R2. The isotope effect (kH/k3H) on 3' carbon-hydrogen bond cleavage is 1.7. This sequence of events is independent of the reductant, consistent with the postulate that C462 is an active site thiol. The C462AR1 has properties similar to C462SR1. Several additional mutant R1s, C230SR1, and C292SR1 were shown to have activities similar to wt-R1 with both TR/TRR/NADPH and DTT.