Electron transfer reactions in Zn-substituted cytochrome P450cam

We have investigated photoinduced electron transfer (ET) reactions between zinc-substituted cytochrome P450cam (ZnP450) and several inorganic reagents by using the laser flash photolysis method, to reveal roles of the electrostatic interactions in the regulation of the ET reactions. The laser pulse irradiation to ZnP450 yielded a strong reductant, the triplet excited state of ZnP450, (3)ZnP450, which was able to transfer one electron to anionic redox partners, OsCl(6)(2-) and Fe(CN)(6)(3-), with formation of the porphyrin pi-cation radical, ZnP450(+). In contrast, the ET reactions from (3)ZnP450 to cationic redox partners, such as Ru(NH(3))(6)(3+) and Co(phen)(3)(3+), were not observed even in the presence of 100-fold excess of the oxidant. One of the possible interpretations for the preferential ET to the anionic redox partner is that the cationic patch on the P450cam surface, a putative interaction site for the anionic reagents, is located near the heme (less than 10 A from the heme edge), while the anionic surface is far from the heme moiety (more than 16 A from the heme edge), which would yield 8000-fold faster ET rates through the cationic patch. The ET rate through the anionic patch to the cationic partner would be substantially slower than that of the phosphorescence process in (3)ZnP450, resulting in no ET reactions to the cationic reagents. These results demonstrate that the asymmetrical charge distribution on the protein surface is critical for the ET reaction in P450cam.     

A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration

A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic NADH regeneration catalyzed by glycerol dehydrogenase (GLD) in Escherichia coli whole cell biocatalysts. Recombinant E. coli harboring the P450cam system, but lacking GLD, exhibited little activity for camphor hydroxylation. In contrast, coexpression of GLD with the proteinaceous electron transfer components of P450cam resulted in about tenfold improvement in the substrate conversion, implying that the whole cell biocatalyst utilized molecular oxygen, endogenous NADH, and glycerol in the cell for catalysis. The addition of glycerol to the reaction media further promoted camphor hydroxylation, suggesting that exogenous glycerol is also available for GLD in the host cell and actively participates in the catalytic cycle. These results clearly show the utility of GLD towards functional reconstruction of the native P450cam system. The present approach may also be useful for E. coli whole cell biocatalysts with the other NADH-dependent oxygenases and oxidoreductases.     

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.     

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.     

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.     

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.     

An anomaly in the resonance Raman spectra of cytochrome P-450cam in the ferrous high-spin state.

Resonance Raman spectra of cytochrome P-450cam (P-450cam) and its enzymatically inactive form (P-420) in various oxidation and spin states were measured for the first time. The Raman spectrum of reduced P-450cam was unusual in the sense that the "oxidation-state marker" appeared at an unexpectedly lower frequency (1346 cm-1) in comparison with those of other reduced hemoproteins (approximately 1355-approximately 1365 cm-1), whereas that of oxidized P-450cam was located at a normal frequency. This anomaly in the Raman spectrum of reduced P-450cam can be explained by assuming electron delocalization from the fifth ligand, presumably a thiolate anion, to the antibonding pi orbital of the porphyrin ring. The corresponding Raman line of reduced P-420 appeared at a normal frequency (1360 cm-1), suggesting a status change or replacement of the fifth ligand upon conversion from P-450cam to P-420. The Raman spectrum of reduced P-450cam-metyrapone complex was very similar to that of ferrous cytochrome b5.     

Clay-bridged electron transfer between cytochrome p450(cam) and electrode

We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450(cam) on a glassy carbon electrode modified with sodium montmorillonite. The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process. The electron transfer rate is in the range from 5 to 152 s(-1) with scan rates from 0.4 to 12 V/s, respectively. These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450(cam). The formal potential of adsorbed P450(cam) is -139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450(cam) in solution. UV-VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450(cam) in solution. However, P450(cam) adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential.     

Spectroelectrochemistry of cytochrome P450cam

The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.     

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.     

L358P mutation on cytochrome P450cam simulates structural changes upon putidaredoxin binding: the structural changes trigger electron transfer to oxy-P450cam from electron donors

To investigate the functional and structural characterization of a crucial cytochrome P450cam (P450cam)-putidaredoxin (Pdx) complex, we utilized a mutant whose spectroscopic property corresponds to the properties of the wild type P450cam in the presence of Pdx. The 1H NMR spectrum of the carbonmonoxy adduct of the mutant, the Leu-358 --> Pro mutant (L358P), in the absence of Pdx showed that the ring current-shifted signals arising from d-camphor were upfield-shifted and observed as resolved signals, which are typical for the wild type enzyme in the presence of Pdx. Signals from the beta-proton of the axial cysteine and the gamma-methyl group of Thr-252 were also shifted upfield and down-field, respectively, in the L358P mutant as observed for Pdx-bound wild type P450cam. The close similarity in the NMR spectra suggests that the heme environment of the L358P mutant mimics that of the Pdx-bound enzyme. The functional analysis of the L358P mutant has revealed that the oxygen adduct of the L358P mutant can promote the oxygenation reaction for d-camphor with nonphysiological electron donors such as dithionite and ascorbic acid, showing that oxygenated L358P is "activated" to receive electron from the donor. Based on the structural and functional characterization of the L358P mutant, we conclude that the Pdx-induced structural changes in P450cam would facilitate the electron transfer from the electron donor, and the Pdx binding to P450cam would be a trigger for the electron transfer to oxygenated P450cam.     

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.     

Crystal structure of P450cin in a complex with its substrate, 1,8-cineole, a close structural homologue to D-camphor, the substrate for P450cam

Cytochrome P450cin catalyzes the monooxygenation of 1,8-cineole, which is structurally very similar to d-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1,8-cineole and d-camphor are C(10) monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 A resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe(2)S(2) redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3.     

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.     

Effect of the tyrosine 96 hydrogen bond on the inactivation of cytochrome P-450cam induced by hydrostatic pressure

The effects of removal of the tyrosine 96 hydrogen bond on the stability and conformational events of cytochrome P-450cam are presented in this communication. Hydrostatic pressure has been used as a tool to perturbe the structure leading to the formation of cytochrome P-420, an inactivated but soluble and undenatured form of the enzyme. We show that the spin transition of cytochrome P-450cam, which is known to be influenced by hydrostatic pressure, is affected by this single mutation. The free energy of stabilisation of native substrate-free cytochrome P-450cam is not affected by the removal of the tyrosine 96 hydrogen bond via mutagenesis to phenylalanine, whereas the substrate-bound protein shows a difference of 21 kJ/mol. These results, as well as an observed 110 ml/mol difference for the volume of the inactivation reaction between substrate-bound native and mutant proteins, have been interpreted in terms of a more hydrated heme pocket for the site-directed mutant at position 96 compared to the wild-type protein where camphor is tightly bound via the tyrosine 96 hydrogen bond and water excluded from the active site.     

Role of protein and substrate dynamics in catalysis by Pseudomonas putida cytochrome P450cam

The role of protein structural flexibility and substrate dynamics in catalysis by cytochrome P450 enzymes is an area of current interest. We have addressed these in cytochrome P450(cam) (P450(cam)) and its Y96A mutant with camphor and its related compounds using fluorescence spectroscopy. Previously [Prasad et al. (2000) FEBS Lett. 477, 157-160], we provided experimental support to dynamic fluctuations in P450(cam), and substrate access into the active site region via the channel next to the flexible F-G helix-loop-helix segment. In the investigation described here, we show that the dynamic fluctuations in the enzyme are substrate dependent as reflected by tryptophan fluorescence quenching experiments. The orientation of tryptophan relative to heme (kappa(2)) for W42 obtained from time-resolved tryptophan fluorescence measurements show variation with type of substrate bound to P450(cam) suggesting regions distant from heme-binding site are affected by physicochemical and steric characteristics/protein-substrate interactions of P450(cam) active site. We monitored substrate dynamics in the active site region of P450(cam) by time-resolved substrate anisotropy measurements. The anisotropy decay of substrates bound to P450(cam) indicate that mobility of substrates is modulated by physicochemical and steric characteristics/protein-substrate interactions of local active site structure, and provides an understanding of factors controlling observed hydroxylated products for substrate bound P450(cam) complexes. The present study shows that P450(cam) local and peripheral structural flexibility and heterogeneity along with substrate mobility play an important role in regulating substrate binding orientation during catalysis and accommodating diverse range of substrates within P450(cam) heme pocket.     

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.     

Continuous B-epitope maps of cytochrome P450cam (CYP101) obtained by peptide scanning: correlation to spatial structure

Protein continuous B-epitopes can be revealed using short synthetic peptides that overlap a known protein sequence. Since the whole protein surface is considered to possess antigenic properties, a question that arises is whether a set of linear B-epitopes determined by peptide scanning correlates with a protein spatial structure. We have chosen cytochrome P450cam (CYP101) of Pseudomonas putida, with known 3D structure, as a template. Sera of two rabbits and antibody egg yolk preparations from three chickens were produced against the P450cam molecule. These polyclonals were analyzed separately in ELISA with 409 overlapping P450cam hexapeptides. The whole set of continuous antigenic sites of P450cam covered about 45% of the P450cam sequence. However, immunodominant sites (those revealed with more than 50% antibody preparations), the so-called "antigenic core," represent only 9% of the protein sequence. While the amount of water-accessible residues in the total antigenic map (42%) was close to that in the whole native P450cam molecule (39%), the amount of water-accessible residues in the antigenic core was significantly higher (64%). These results led to the conclusion that antigenic core epitopes can be associated to the molecular surface, whereas epitopes with low detection frequency may partly correspond to unfolded regions of the protein molecule.     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

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.