Phthalate dioxygenase reductase (PDR) is the electron transferase component of the phthalate dioxygenase system. It is a modular enzyme consisting of distinct iron-sulfur, flavin mononucleotide (FMN) and pyridine nucleotide binding domains. We have taken advantage of this modularity and removed the 10-kDa iron-sulfur domain by selective proteolytic cleavage between residues N229 and T230 in a solvent-accessible peptide that links the iron-sulfur and pyridine nucleotide binding domains. The resulting PDR(-FeS) has a molecular weight of 25,792 +/- 10 and the same amino terminus as the native PDR. It has spectral features that are very similar to the flavin component of the PDR absorbance spectrum. Remarkably, despite the magnitude of this structural modification, the kinetic, redox, and pyridine nucleotide binding properties of PDR(-FeS) are very similar to those reported for PDR [Gassner, G., et al. (1994) Biochemistry 33, 12184-12193]. The principal mechanistic feature distinguishing PDR(-FeS) from PDR is the inability of the attenuated enzyme to carry out intramolecular electron transfer. The reaction of PDR(-FeS) with NADH consists of binding and the formation of an initial Michaelis complex (MC-1') (Kd approximately 25 microM), isomerization of the enzyme (120 s-1) to form a charge-transfer complex with FMN (CT-1'), hydride transfer to the FMN (76 s-1) with formation of a second charge-transfer complex (CT*'), and finally release of nicotinamide adenine dinucleotide (NAD) (58 s-1) from the reduced enzyme. The rate of NAD release from PDR(-FeS) is nearly the same as the rate of NAD release and intramolecular electron transfer in the reductive half-reaction of PDR, which supports the idea that the release of NAD triggers intramolecular electron transfer in PDR. The midpoint potential of the oxidized/semiquinone couple of PDR(-FeS) (-170 mV) is the same as the value measured for PDR. A value of -235 mV is measured for the midpoint potential of the semiquinone/hydroquinone couple of PDR(-FeS), which is approximately 50 mV more positive than the PDR2e-/PDR3e- redox couple at pH 7. NAD binds to PDR(-FeS) about 20-fold more weakly than does NADH; the enzyme redox state has no significant influence on pyridine nucleotide binding affinity.