Activity profile of glutathione-dependent enzymes and respiratory chain complexes in rats supplemented with antioxidants and treated with carcinogens

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated. It was found that Pd/P450cam complex formation and decay obeys biphasic kinetics in contrast to the monophasic one for complexes formed by other redox partners within the system. Evidence for PdR/P450cam complex formation was obtained. It was found that, in contrast to Pd, which binds only to its redox partners, PdR and P450cam were able to form PdR/PdR and P450cam/P450cam complexes. A ternary PdR/Pd/P450cam complex was also registered. Its lifetime was sufficient to permit up to 60 turnovers to occur. The binding of Pd to P450cam and to PdR within the ternary complex occurred at distinct sites, with Pd serving as a bridge between the two proteins.     

Structural biology of redox partner interactions in P450cam monooxygenase: A fresh look at an old system

The P450cam monooxygenase system consists of three separate proteins: the FAD-containing, NADH-dependent oxidoreductase (putidaredoxin reductase or Pdr), cytochrome P450cam and the 2Fe2S ferredoxin (putidaredoxin or Pdx), which transfers electrons from Pdr to P450cam. Over the past few years our lab has focused on the interaction between these redox components. It has been known for some time that Pdx can serve as an effector in addition to its electron shuttle role. The binding of Pdx to P450cam is thought to induce structural changes in the P450cam active site that couple electron transfer to substrate hydroxylation. The nature of these structural changes has remained unclear until a particular mutant of P450cam (Leu358Pro) was found to exhibit spectral perturbations similar to those observed in wild type P450cam bound to Pdx. The crystal structure of the L358P variant has provided some important insights on what might be happening when Pdx docks. In addition to these studies, many Pdx mutants have been analyzed to identify regions important for electron transfer. Somewhat surprisingly, we found that Pdx residues predicted to be at the P450cam–Pdx interface play different roles in the reduction of ferric P450cam and the ferrous P450–O₂ complex. More recently we have succeeded in obtaining the structure of a chemically cross-linked Pdr–Pdx complex. This fusion protein represents a valid model for the noncovalent Pdr–Pdx complex as it retains the redox activities of native Pdr and Pdx and supports monooxygenase reactions catalyzed by P450cam. The insights gained from these studies will be summarized in this review.     

Atomic force microscopy detection of molecular complexes in multiprotein P450cam containing monooxygenase system

The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm.     

The role of water molecules in the association of cytochrome P450cam with putidaredoxin. An osmotic pressure study

We have investigated the osmotic pressure dependence of the association between ferric cytochrome P450cam and putidaredoxin (Pdx) to gain an insight into the role of water molecules in the P450cam-reduced Pdx complexation amenable to physiological electron transfer. The association constant was evaluated from the electron transfer rates from reduced Pdx to P450cam. The natural logarithm of the association constant K(a) was linearly reduced by the osmotic pressure, and osmotic stress yields uptake of 25 waters upon association. In contrast, uptake of only 13 waters is observed from the osmotic pressure dependence of the association in the nonphysiological redox partners P450cam and oxidized Pdx. Although general protein-protein associations proceed through dehydration around the complex interface, the interfacial waters could mediate hydrogen-bonding interactions. Therefore, about 10 more interfacial waters imply an additional water-mediated hydrogen-bonding network in the P450cam.reduced Pdx complex, which does not exist in the complex with oxidized Pdx. It is also possible that the water-mediated hydrogen-bonding interactions support a high P450cam affinity for reduced (K(a) = 0.83 microm(-1)) relative to oxidized (K(a) = 0.058 microm(-1)) Pdx. This study points to a novel role of solvents in assisting redox state-dependent interaction between P450cam and Pdx.     

Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.     

Cytochrome P-450cam and putidaredoxin interaction during electron transfer.

Cytochrome P-450cam, the bacterial hemeprotein which catalyzes the 5-exo-hydroxylation of d-camphor, requires two electrons to activate molecular oxygen for this monooxygenase reaction. These two electrons are transferred to cytochrome P-450cam in two one-electron steps by the physiological reductant, putidaredoxin. The present study of the kinetics of reduction of cytochrome P-450cam by reduced putidaredoxin has shown that the reaction obeys first order kinetics with a rate constant of 33 s-1 at 25 degrees C with respect to: 1) the appearance of the carbon monoxide complex of Fe(II) cytochrome P-450cam; 2) the disappearance of the 645 nm absorbance band of high-spin Fe(III) cytochrome P-450cam; and 3) the disappearance of the g = 1.94 EPR signal of reduced putidaredoxin. This data was interpreted as indicative of the rapid formation of a bimolecular complex between reduced putidaredoxin Fe(III) cytochrome P-450cam. The existence of the complex was first shown indirectly by kinetic analysis and secondly directly by electron paramagnetic resonance spectroscopic analysis of samples which were freeze-quenched approximately 16 ms after mixing. The direct evidence for complex formation was the loss of the EPR signal of Fe(III) cytochrome P-450cam upon formation of the complex while the EPR signal of reduced putidaredoxin decays with the same kinetics as the appearance of Fe(II) cytochrome P-450. The mechanism of the loss of the EPR signal of cytochrome P-450 upon formation of the complex is not apparent at this time but may involve a conformational change of cytochrome P-450cam following complex formation.     

Putidaredoxin-to-cytochrome P450cam electron transfer: differences between the two reductive steps required for catalysis

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 30 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 delta106 mutants exhibited considerably higher Kd 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%. On the basis of 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.     

The reduction of putidaredoxin reductase by reduced pyridine nucleotides

Putidaredoxin reductase (PdR), an FAD-containing protein, mediates the transfer of electrons from NADH to putidaredoxin in the cytochrome P-450cam-dependent oxidation of camphor. Using stopped-flow spectrophotometry, reduction of putidaredoxin reductase by NADH (70 microM) at 4 degrees C appeared to be a pseudo-first-order process with a rate constant in excess of 600 s-1. The reduction of putidaredoxin reductase by NADPH was much slower with a second-order rate constant of 530 s-1 M-1 at 4 degrees C. The reduction of the enzyme was monitored at several wavelengths: 455 nm to follow flavin reduction; 700 nm to follow the appearance of the long-wavelength charge-transfer complex; and 513 nm to detect the presence of a semiquinone form of the flavoprotein. There was no apparent semiquinone formation observed during reduction. The charge-transfer complex can be formed in the presence of NAD+, whereas, no charge-transfer band could be detected when PdR was reduced with NADPH. The titration of chemically or NADPH-reduced putidaredoxin reductase with either a stoichiometric or an excess amount of NAD+ resulted in the formation of a charge-transfer complex, indicating that the reduced form of PdR has a high affinity for NAD+ regardless of the method of reduction. The data presented indicate that putidaredoxin reductase is reduced without the formation of semiquinone intermediate and, upon reduction, forms a tight complex with NAD+. The Keq for the reduction of PdR by NADPH is 1.1 and the midpoint potential for this reaction is -317 +/- 5 mV.     

Putidaredoxin-cytochrome p450cam interaction. Spin state of the heme iron modulates putidaredoxin structure

During the monooxygenase reaction catalyzed by cytochrome P450cam (P450cam), a ternary complex of P450cam, reduced putidaredoxin, and d-camphor is formed as an obligatory reaction intermediate. When ligands such as CO, NO, and O2 bind to the heme iron of P450cam in the intermediate complex, the EPR spectrum of reduced putidaredoxin with a characteristic signal at 346 millitesla at 77 K changed into a spectrum having a new signal at 348 millitesla. The experiment with O2 was carried out by employing a mutant P450cam with Asp251 --> Asn or Gly where the rate of electron transfer from putidaredoxin to oxyferrous P450cam is considerably reduced. Such a ligand-induced EPR spectral change of putidaredoxin was also shown in situ in Pseudomonas putida. Mutations introduced into the neighborhood of the iron-sulfur cluster of putidaredoxin revealed that a Ser44 --> Gly mutation mimicked the ligand-induced spectral change of putidaredoxin. Arg109 and Arg112, which are in the putative putidaredoxin binding site of P450cam, were essential for the spectral changes of putidaredoxin in the complex. These results indicate that a change in the P450cam active site that is the consequence of an altered spin state is transmitted to putidaredoxin within the ternary complex and produces a conformational change of the 2Fe-2S active center.     

Laser flash induced electron transfer in P450cam monooxygenase: putidaredoxin reductase-putidaredoxin interaction

The P450cam monooxygenase from Pseudomonas putida consists of three redox proteins: NADH-putidaredoxin reductase (Pdr), putidaredoxin (Pdx), and cytochrome P450cam. The redox properties of the FAD-containing Pdr and the mechanism of Pdr-Pdx complex formation are the least studied aspects of this system. We have utilized laser flash photolysis techniques to produce the one-electron-reduced species of Pdr, to characterize its spectral and electron-transferring properties, and to investigate the mechanism of its interaction with Pdx. Upon flash-induced reduction by 5-deazariboflavin semiquinone, the flavoprotein forms a blue neutral FAD semiquinone (FADH(*)). The FAD semiquinone was unstable and partially disproportionated into fully oxidized and fully reduced flavin. The rate of FADH(*) decay was dependent on ionic strength and NAD(+). In the mixture of Pdr and Pdx, where the flavoprotein was present in excess, electron transfer (ET) from FADH(*) to the iron-sulfur cluster was observed. The Pdr-to-Pdx ET rates were maximal at an ionic strength of 0.35 where a kinetic dissociation constant (K(d)) for the transient Pdr-Pdx complex and a limiting k(obs) value were equal to 5 microM and 226 s(-1), respectively. This indicates that FADH(*) is a kinetically significant intermediate in the turnover of P450cam monooxygenase. Transient kinetics as a function of ionic strength suggest that, in contrast to the Pdx-P450cam redox couple where complex formation is predominantly electrostatic, the Pdx-Pdr association is driven by nonelectrostatic interactions.     

Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the flavin adenine dinucleotide (FAD/FADH2) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD+/NADH) and donating them in two separate, one-electron-transfer steps to the iron-sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin-NADP+ reductase (FNR), has no other active redox moieties (e.g., sulfhydryl groups) and can exist in three different oxidation states: (i) oxidized quinone, (ii) one-electron reduced semiquinone (stable neutral species (blue) or unstable radical anion (red)), and (iii) two-electron fully reduced hydroquinone. Here, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of equilibria among the redox components in this initial electron-transfer step of the P450 cycle. A spectroelectrochemical technique was used to measure the midpoint oxidation-reduction potential of PdR that had been carefully purified of all residual NAD+, E0' = -369 +/- 10 mV at pH 7.6, which is more negative than previously reported and more negative than the pyridine nucleotide NADH/NAD+ (-330 mV). After addition of NAD+, the formation of the oxidized reductase-oxidized pyridine nucleotide complex was followed by the two-electron-transfer redox reaction, PdRox:NAD+ + 2e- --> PdRrd:NAD+, when the electrode potential was lowered. The midpoint potential was a hyperbolic function of increasing NAD+ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be E0' = -230 +/- 10 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH. This thermodynamic control of electron transfer is a shared mechanistic feature with the adrenodoxin P450 and photosynthetic electron-transfer systems but is different from the kinetic control mechanisms in the microsomal P450 systems where multiple reaction pathways draw on reducing power held by NADPH-cytochrome P450 reductase. The redox measurements were combined with protein fluorescence quenching of NAD+ binding to oxidized PdR to establish that the PdRox:NAD+ complex (KD = 230 microM) is about 5 orders of magnitude weaker than PdRrd:NAD+ binding. These results are integrated with known structural and kinetic information for PdR, as well as for AdR and FNR, in support of a compulsory ordered pathway to describe the electron-transfer processes catalyzed by all three reductases.     

Mechanism of O2 activation by cytochrome P450cam studied by isotope effects and transient state kinetics

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.     

A scanning tunneling microscopy (STM) investigation of complex formation between cytochrome P450(cam) and putidaredoxin

We have previously reported the scanning tunnelling microscopy (STM) imaging under buffer of the heme monooxygenase cytochrome P450(cam) from Pseudomonas putida [Faraday Discuss. 116 (2000) 1]. We describe here the adsorption and STM imaging under buffer of complexes of a mutant of cytochrome P450(cam), K344C, and wild-type putidaredoxin (Pdx) on gold(111). The images of Pdx on its own on gold(111) are not uniform, presumably due to multiple orientations of protein adsorption because of the presence of five or more cysteines on the protein surface. STM imaging of a 1:1 mixture of P450(cam)-K344C/Pdx showed a regular array of pairs of different-sized proteins 20-25 A apart arranged in rows across the gold(111) surface which we attribute to the P450(cam)/Pdx complex. The images of the pairs are more regular than those of Pdx on its own, probably as a result of complex formation with P450(cam) partly overcoming the heterogeneity of Pdx adsorption. As far as we are aware this is the first report of STM imaging of a protein/protein complex, and the first direct observation of P450(cam)/Pdx complex formation which is a key step in the catalytic cycle of P450(cam) catalysis. The redox centers of the two proteins are ca. 20 A apart, too far for rapid intracomplex electron transfer. Whether the observed complex is competent for electron transfer or physiologically relevant is not known, and further work is in progress to elucidate the protein-protein interaction.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Molecular mechanism of the electron transfer reaction in cytochrome P450(cam)--putidaredoxin: roles of glutamine 360 at the heme proximal site

We characterized electron transfer (ET) from putidaredoxin (Pdx) to the mutants of cytochrome P450(cam) (P450(cam)), in which one of the residues located on the putative binding site to Pdx, Gln360, was replaced with Glu, Lys, and Leu. The kinetic analysis of the ET reactions from reduced Pdx to ferric P450(cam) (the first ET) and to ferrous oxygenated P450(cam) (the second ET) showed the dissociation constants (K(m)) that were moderately perturbed for the Lys and Leu mutants and the distinctly increased for the Glu mutant. Although the alterations in K(m) indicate that Gln360 is located at the Pdx binding site, the effects of the Gln360 mutations (0.66-20-fold of that of wild type) are smaller than those of the Arg112 mutants (25-2500-fold of that of wild type) [Unno, M., et al. (1996) J. Biol. Chem. 271, 17869-17874], allowing us to conclude that Gln360 much less contributes to the complexation with Pdx than Arg112. The first ET rate (35 s(-1) for wild-type P450(cam)) was substantially reduced in the Glu mutant (5.4 s(-1)), while less perturbation was observed for the Lys (53 s(-1)) and Leu (23 s(-1)) mutants. In the second ET reaction, the retarded ET rate was detected only in the Glu mutant but not in the Lys and Leu mutants. These results showed the smaller mutational effects of Gln360 on the ET reactions than those of the Arg112 mutants. In contrast to the moderate perturbations in the kinetic parameters, the mutations at Gln360 significantly affected both the standard enthalpy and entropy of the redox reaction of P450(cam), which cause the negative shift of the redox potentials for the Fe(3+)/Fe(2+) couple by 20-70 mV. Since the amide group of Gln360 is located near the carbonyl oxygen of the amide group of the axial cysteine, it is plausible that the mutation at Gln360 perturbs the electronic interaction of the axial ligand with heme iron, resulting in the reduction of the redox potentials. We, therefore, conclude that Gln360 primarily regulates the ET reaction of P450(cam) by modulating the redox potential of the heme iron and not by the specific interaction with Pdx or the formation of the ET pathway that are proposed as the regulation mechanism of Arg112.     

Direct observation of a novel perturbed oxyferrous catalytic intermediate during reduced putidaredoxin-initiated turnover of cytochrome P-450-CAM: probing the effector role of putidaredoxin in catalysis

The single turnover of (1R)(+)-camphor-bound oxyferrous cytochrome P450-CAM with one equivalent of dithionite-reduced putidaredoxin (Pdx) was monitored for the appearance of transient intermediates at 3 degrees C by double mixing rapid scanning stopped-flow spectroscopy. With excess camphor, three successive species were observed after generating oxyferrous P450-CAM and reacting versus reduced Pdx: a perturbed oxyferrous derivative, a species that was a mixture of high and low spin Fe(III), and high spin ferric camphor-bound enzyme. The rates of the first two steps, approximately 140 and approximately 85 s(-1), were assigned to formation of the perturbed oxyferrous intermediate and to electron transfer from reduced Pdx, respectively. In the presence of stoichiometric substrate, three phases with similar rates were seen even though the final state is low spin ferric P450-CAM. This is consistent with substrate being hydroxylated during the reaction. The single turnover reaction initiated by adding dioxygen to a preformed reduced P450-CAM.Pdx complex with excess camphor also led to phases with similar rates. It is proposed that formation of the perturbed oxyferrous intermediate reflects alteration of H-bonding to the proximal Cys, increasing the reduction potential of the oxyferrous state and triggering electron transfer from reduced Pdx. This species may be a direct spectral signature of the effector role of Pdx on P450-CAM reactivity (i.e. during catalysis). The substrate-free oxyferrous enzyme also reacted readily with reduced Pdx, showing that the inability of substrate-free P450-CAM to accept electrons from reduced Pdx and function as an NADH oxidase is completely due to the incapacity of reduced Pdx to deliver the first but not the second electron.     

Molecular dynamics of the P450cam–Pdx complex reveals complex stability and novel interface contacts

Cytochrome P450cam catalyzes the stereo and regiospecific hydroxylation of camphor to 5‐exo‐hydroxylcamphor. The two electrons for the oxidation of camphor are provided by putidaredoxin (Pdx), a Fe₂S₂ containing protein. Two recent crystal structures of the P450cam–Pdx complex, one solved with the aid of covalent cross‐linking and one without, have provided a structural picture of the redox partner interaction. To study the stability of the complex structure and the minor differences between the recent crystal structures, a 100 nanosecond molecular dynamics (MD) simulation of the cross‐linked structure, mutated in silico to wild type and the linker molecule removed, was performed. The complex was stable over the course of the simulation though conformational changes including the movement of the C helix of P450cam further toward Pdx allowed for the formation of a number of new contacts at the complex interface that remained stable throughout the simulation. While several minor crystal contacts were lost in the simulation, all major contacts that had been experimentally studied previously were maintained. The equilibrated MD structure contained a mixture of contacts resembling both the cross‐linked and noncovalent structures and the newly identified interactions. Finally, the reformation of the P450cam Asp251–Arg186 ion pair in the MD simulation mirrors the ion pair observed in the more promiscuous CYP101D1 and suggests that the Asp251–Arg186 ion pair may be important.     

Molecular Characterization of a Class I P450 Electron Transfer System from Novosphingobium aromaticivorans DSM12444

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from the oligotrophic bacterium Novosphingobium aromaticivorans DSM12444 are heme monooxygenases that receive electrons from NADH via Arx, a [2Fe-2S] ferredoxin, and ArR, a ferredoxin reductase. These systems show fast NADH turnovers (k_cat = 39–91 s⁻¹) that are efficiently coupled to product formation. The three-dimensional structures of ArR, Arx, and CYP101D1, which form a physiological class I P450 electron transfer chain, have been resolved by x-ray crystallography. The general structural features of these proteins are similar to their counterparts in other class I systems such as putidaredoxin reductase (PdR), putidaredoxin (Pdx), and CYP101A1 of the camphor hydroxylase system from Pseudomonas putida, and adrenodoxin (Adx) of the mitochondrial steroidogenic CYP11 and CYP24A1 systems. However, significant differences in the proposed protein-protein interaction surfaces of the ferredoxin reductase, ferredoxin, and P450 enzyme are found. There are regions of positive charge on the likely interaction face of ArR and CYP101D1 and a corresponding negatively charged area on the surface of Arx. The [2Fe-2S] cluster binding loop in Arx also has a neutral, hydrophobic patch on the surface. These surface characteristics are more in common with those of Adx than Pdx. The observed structural features are consistent with the ionic strength dependence of the activity.     

Complex formation of cytochrome P450cam with Putidaredoxin. Evidence for protein-specific interactions involving the proximal thiolate ligand.

We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam). These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.     

Electron transport in cytochromes P-450 by covalent switching.

The mechanism of electron transfer in cytochrome P-450cam is presented in terms of a covalent switching mechanism. We present a model of putidaredoxin built by homology, which helps explain protein-protein interactions. The mechanism is general enough to account for the genetic variations found in the superfamily of cytochromes P-450. The detail should assist in the design of novel P-450 inhibitors and may have wider implications. The sequence analysis supports our protein model, and highlights the role of cystein and aromatic residues in electron-transport mechanisms. Eukaryotic cytochromes P-450 appear to have evolved their own intramolecular tryptophan electron-transfer mediator, unlike prokaryotic P. putida P-450cam, which still relies upon the C-terminal tryptophan of its attendant electron-transport protein, putidaredoxin. On this basis our protein model is capable of rationalizing the transfer of electrons from NADH to the active site of P-450. At the electronic level the covalent switching that transfers pairs of electrons not only provides a plausible mechanism, but may also have ramifications in a wider context.