Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from Pseudomonas putida was studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and In the three-component camphor hydroxylase system from Pseudomonas putida, a FAD-containing NADH-putidaredoxin reductase (Pdr) 1 receives two electrons as a hydride from NADH and delivers these as single reducing equivalents to two molecules of a [2Fe-2S] ferredoxin, putidaredoxin (Pdx). Two molecules of Pdx, in turn, donate electrons to one molecule of P450cam that oxidizes D-camphor to 5-exo-hydroxycamphor using molecular oxygen (1). Reactions of NADH oxidation/Pdx reduction and Pdx oxidation/camphor hydroxylation are highly coupled and proceed with turnover numbers of 16,000 and 2,000 min Ϫ1 , respectively (2, 3). To provide efficient catalytic turnover of P450cam monooxygenase, Pdx must form productive transient or long lived electron transfer complexes with its redox partners. To date, there is more supporting evidence for the electron transfer shuttle mechanism in the P450cam monooxygenase, according to which Pdx acts as a freely diffusible shuttle between Pdr and P450cam (4 -7), as opposed to the mechanism that requires formation of a ternary complex between the three redox partners (8, 9).Determination of the x-ray and NMR structures of P450cam (10) and Pdx (11), respectively, has enabled a better understanding structure-function relations in these proteins. The lack of structural information on Pdr, however, was the primary reason that the Pdr-Pdx redox couple was the least studied in this system. The reported data are contradictory and suggest that steric, electrostatic, or hydrophobic components are involved in the association between the flavo-and iron-sulfur proteins (5,(12)(13)(14)(15). Investigation of the Pdr-Pdx interaction is necessary for unraveling the mechanism of the P450cam and other homologous three component monooxygenase systems. The long range interprotein electron transfer is a fundamental process, and, thus, elucidation of specific interactions that assist and stabilize PdrPdx complex and mediate FAD-to-[2Fe-2S] electron transfer is of fundamental importance.Recently determined x-ray structures of oxidized Pdr and oxidized and reduced Pdx have provided a structural base for studying electron transfer and molecular recognition in the Pdr-Pdx redox couple (16 -18). In this study, we utilized computer graphics techniques and crystal structures of the proteins to develop three-dimensional models for the Pdr-Pdx complex. To test the validity of the proposed models and to further investigate structure-function relations in Pdx, four residues of the iron-sulfur protein located at the protein-protein interface in the model complexes were mutated, and the redox prop...