Cytochrome P450cam (P450cam) is the terminal monooxygenase in a three-component camphor hydroxylating system from Pseudomonas putida. The reaction cycle requires two distinct electron transfer (ET) processes from the [2Fe-2S] containing putidaredoxin (Pdx) to P450cam. Even though the mechanism of interaction and ET between the two proteins has been under investigation for over thirty years, the second reductive step and the effector role of Pdx are not fully understood. We utilized mutagenesis, kinetic, and computer modeling approaches to better understand differences between the two Pdx-to-P450cam ET events. Our results indicate that interacting residues and the ET pathways in the complexes formed between reduced Pdx (Pdx r ) and the ferric and ferrous dioxygen-bound forms of P450cam (oxy-P450cam) are different. Pdx Asp38 and Trp106 were found to be key players in both reductive steps. Compared to the wild type Pdx, the D38A, W106A, and Δ106 mutants exhibited considerably higher K d values for ferric P450cam and retained ca. 20% of the first electron transferring ability. In contrast, the binding affinity of the mutants for oxy-P450cam was not substantially altered while the second ET rates were <1%. Based on the kinetic and modeling data we conclude that (i) P450cam-Pdx interaction is highly specific in part because it is guided/ controlled by the redox state of both partners; (ii) there are alternative ET routes from Pdx r to ferric P450cam and a unique pathway to oxy-P450cam involving Asp38; (iii) Pdx Trp106 is a key structural element that couples the second ET event to product formation possibly via its "push" effect on the heme binding loop.Cytochromes P450 (P450) participate in a variety of metabolic processes and catalyze the monooxygenation of a wide range of aromatic and aliphatic substrates. Mixed function oxidation reactions catalyzed by P450s (Fig. 1) require an input of two electrons that originate from NAD(P)H and are supplied by redox-linked proteins. The most extensively characterized P450, a camphor hydroxylating cytochrome P450cam (P450cam) from Pseudomonas putida, receives reducing equivalents from a [2Fe-2S] ferredoxin, putidaredoxin (Pdx), which shuttles between the hemoprotein and a FAD-containing putidaredoxin reductase (Pdr) (1). Proteinprotein interactions in P450cam monooxygenase are highly specific and neither Pdx nor Pdr † This research was supported by National Institute of Health Grants GM67637 (to I.F.S.) and GM33688 (to T.L.P.). *To whom correspondence should be addressed: Tel: 949-824-1953, Fax: 949-824-3280, E-mail: sevrioui@uci.edu is functionally interchangeable with the homologous proteins from other redox systems (2-4). The strict requirement of Pdx for reduction of ferrous dioxygen-bound P450cam (oxyP450cam) and product formation is thought to be due to its unique ability to couple electron flow to successful substrate turnover (3,5,6). Owing to technical difficulties in monitoring oxyP450cam reduction, this process remains the least studied in the P450cam catalyt...

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...

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The application of atomic force microscopy (AFM) to the identification and visualization of individual molecules and their complexes in a reconstituted monooxygenase P450 2B4 system without the phospholipid was demonstrated. The method employed in this study distinguishes the monomeric proteins from their binary complexes and, also, the binary from the ternary complexes. The AFM images of the full-length P450 2B4 system's constituent components - cytochrome P450 2B4 (2B4), NADPH-cytochrome P450 reductase and cytochrome b5 (b5), were obtained on highly-oriented pyrolitic graphite. The typical heights of the d-2B4, d-flavoprotein (Fp) and d-b5 molecules were measured and found to be 2.2 +/- 0.2, 2.3 +/- 0.2 and 1.8 +/- 0.1 nm, respectively. The measured heights of the binary d-Fp/d-2B4 and d-2B4/d-b5 complexes were estimated to be 3.4 +/- 0.2 and 2.8 +/- 0.2 nm, respectively. No formation of d-Fp/d-b5 complexes was registered. The ternary d-Fp/d-2B4/d-b5 complexes were visualized and their heights were found to be roughly equal to 4.3 +/- 0.3 nm and 6.2 +/- 0.3 nm.

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