The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450 cam (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed in Escherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450 cam system was measured, and kinetic parameters were determined. Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner. Some mutations altered interactions of Pdx with one redox partner but not the other. Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101. Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101.Multiprotein redox enzyme systems such as methane monooxygenase, cytochrome P450s, and diooxygenases of similar molecular architecture are being investigated as biocatalysts for conversion of organic substrates with no functional groups into oxygen-bearing compounds with high regio-or stereo-selectivity. Maximizing the catalytic efficiency of such systems requires knowledge of the pathways of electron transfer and of the surface regions and amino acid residues involved in the interaction of the redox partner subunits.Cytochrome P450 cam (CYP101) has been intensively investigated for over 20 years as a model P450 system (1). This soluble P450 (from Pseudomonas putida) consists of three subunits: putidaredoxin reductase (PdR, 1 M r Ϸ 43,500), putidaredoxin (Pdx, M r Ϸ 11,600), and cytochrome P450 cam hydroxylase (CYP101, M r Ϸ 45,000). The genes, camA (PdR), camB (Pdx), and camC (CYP101) from the cam operon have been cloned and sequenced, and the protein subunits were expressed in individual clones (2-5). Structural information is available for two of the three subunits of the CYP101 system. CYP101 has been crystallized in a number of states, and the structure is well defined (6 -8). Structural information on Pdx comes from solution 1 H NMR studies by Pochapsky and co-workers (9 -11), and they have proposed a model.Electron transfer in this system proceeds from NADH via the flavin group of PdR to the 2Fe-2S center of Pdx and then to the heme iron of CYP101 which accepts one electron at a time from Pdx. Because the details of the electron transfer pathway from one subunit to the next are missing, it is not known exactly how the subunits bind for the transfer of electrons. Ionic strength is well known to have an effect on binding and electron transfer suggesting that salt bridges are important in these interactions (12, 13). The role of some amino acid residues, specifically Trp-106 on Pdx and Arg-112 on CYP101, is known to be important for binding and electron transfer (14 -18). Residues involved in the PdR-Pdx interaction are not necessarily the same as tho...

The large potential of redox enzymes to carry out formation of high value organic compounds motivates the search for innovative strategies to regenerate the cofactors needed by their biocatalytic cycles. Here, we describe a bioreactor where the reducing power to the cycle is supplied directly to purified cytochrome CYP101 (P450cam; EC 1.14.15.1) through its natural redox partner (putidaredoxin) using an antimony-doped tin oxide working electrode. Required oxygen was produced at a Pt counter electrode by water electrolysis. A continuous catalytic cycle was sustained for more than 5 h and 2,600 enzyme turnovers. The maximum product formation rate was 36 nmol of 5-exo-hydroxycamphor͞nmol of CYP101 per min.

We present a detailed atomic level view of the interactions between cytochrome P450cam (CYP101) and its natural redox partner, putidaredoxin (Pdx). A combined theoretical (Poisson-Boltzmann electrostatic calculations, electron transfer pathways, and molecular dynamics) and experimental (site-directed mutagenesis and kinetic analysis) study is used to pinpoint surface residues in both proteins that are important for electron transfer, binding, or both. We find a situation where the electrostatically complementary regions at the surface of both proteins overlap strongly with regions that have large electron transfer couplings to the redox centers. This means that a small surface patch in each protein is involved in binding and electron transfer. A dominant electron transfer pathway is identified, corresponding to an electron leaving the reduced Fe 2 S 2 in Pdx, going through Cys39 and Asp38, and transferring across the interprotein interface to Arg112 (CYP101), then to a heme propionate group, and finally to the heme iron center.

The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T1 and T2 values and 1H-15N NOEs have been measured for the diamagnetic region of the protein. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameters (S2), the effective correlation time for internal motions (tau e), and the 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (tau(m)). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on the microsecond to millisecond time scale for the oxidized Pdx, in comparison with the reduced Pdx. These results clearly indicate that the oxidized protein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proton exchange. In addition, we observed very high T1/T2 ratios for residues 33 and 34, giving rise to a large Rex contribution. Residue 34 is believed to be involved in the binding of Pdx to cytochrome P450cam (CYP101). The differences in the backbone dynamics are discussed in relation to the oxidation states of Pdx, and their impact on electron transfer. The entropy change occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.

The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4-hydroxy benzoate (4HB) for the ubiquinone pathway. In Escherichia coli, CL is monomeric, with 164 residues. We have determined the structure of the CL product complex by crystallographic heavy-atom methods and report the structure at 1.4-A resolution for a fully active double Cys-to-Ser mutant and at 2.0-A resolution for the wild-type. The fold involves a 6-stranded antiparallel beta-sheet with no spanning helices and novel connectivity. The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main-chain amides and by the buried side-chains of Arg 76 and Glu 155. The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix-turn-helix loops. The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10-fold stronger binding of product than substrate. Substrate binding and kinetically rate-limiting product release apparently require the rearrangement of these active-site-covering loops. Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane-bound enzyme 4HB octaprenyltransferase.