Reduced flavodoxin I (Fld1) is required in Escherichia coli for reductive radical generation in AdoMet-dependent radical enzymes and reductive activation of cobalamin-dependent methionine synthase. Ferredoxin (Fd) and flavodoxin II (Fld2) are also present, although their precise roles have not been ascertained. Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) was discovered in E. coli as an NADPH-dependent reductant of Fld1 that facilitated generation of active methionine synthase in vitro; FNR and Fld1 will also supply electrons for the reductive cleavage of AdoMet essential for generating protein or substrate radicals in pyruvate formate-lyase, class III ribonucleotide reductase, biotin synthase, and, potentially, lipoyl synthase. As part of ongoing efforts to understand the various redox pathways that will support AdoMet-dependent radical enzymes in E. coli, we have examined the relative specificity of E. coli FNR for Fd, Fld1, and Fld2. While FNR will reduce all three proteins, Fd is the kinetically and thermodynamically preferred partner. Fd binds to FNR with high affinity (K(d)<or=0.5 microM) and is reduced under single-turnover conditions with k(obs)=2.3s(-1) and under steady state conditions with k(cat)=0.15s(-1). Fld1 and Fld2 behave similarly with respect to FNR, with affinities approximately 4- to 7-fold weaker and reduction rates that are 10- to 100-fold slower than those for Fd. Surprisingly we find that Fld1 and Fld2 can obtain electrons from reduced Fd at rates that are comparable to those obtained with reduced FNR. Thus we propose that the primary electron acceptor for E. coli FNR is Fd, while Fld1 can obtain electrons slowly either from FNR or via Fd as a mediator.