Escherichia coli flavorubredoxin (FlRd) belongs to the family of flavodiiron proteins (FDPs), microbial enzymes that are expressed to scavenge nitric oxide (NO) under anaerobic conditions. To degrade NO, FlRd has to be reduced by NADH via the FAD-binding protein flavorubredoxin reductase, thus the kinetics of electron transfer along this pathway was investigated by stopped-flow absorption spectroscopy. We found that NADH, but not NADPH, quickly reduces the FlRd-reductase (k = 5.5 +/- 2.2 x 10(6) M(-1).s(-1) at 5 degrees C), with a limiting rate of 255 +/- 17 s(-1). The reductase in turn quickly reduces the rubredoxin (Rd) center of FlRd, as assessed at 5 degrees C working with the native FlRd enzyme (k = 2.4 +/- 0.1 x 10(6) m(-1).s(-1)) and with its isolated Rd-domain (k approximately 1 x 10(7) M(-1).s(-1)); in both cases the reaction was found to be dependent on pH and ionic strength. In FlRd the fast reduction of the Rd center occurs synchronously with the formation of flavin mononucleotide semiquinone. Our data provide evidence that (a) FlRd-reductase rapidly shuttles electrons between NADH and FlRd, a prerequisite for NO reduction in this detoxification pathway, and (b) the electron accepting site in FlRd, the Rd center, is in very fast redox equilibrium with the flavin mononucleotide.