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 competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex

Cytochrome b5 has been genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450cam from Pseudomonas putida [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. In the mutant cytochrome b5, threonine is replaced by a cysteine at position 65 (T65C) and has been labeled with the environmentally sensitive fluorophore acrylodan. In this paper, the physiological P-450cam reductant putidaredoxin, an Fe2S2.Cys4 iron-sulfur protein, is shown to competitively inhibit the cytochrome b5 association, suggesting that cytochrome b5 and putidaredoxin bind to a similar site on the cytochrome P-450cam surface. Since the crystal structures for both cytochrome b5 and cytochrome P-450cam have been solved to high resolution, the complex has been computer modeled, and a good fit was found on the proximal surface of nearest approach to the P-450cam heme prosthetic group. The proposed model includes electrostatic contacts between conserved cytochrome b5 carboxylates Glu-44, Glu-48, Asp-60, and the exposed heme propionate with cytochrome P-450cam basic residues Lys-344, Arg-72, Arg-112, and Arg-364, respectively. Putidaredoxin has similarly been shown to contain a carboxylate-based binding surface, and the current results suggest that if the model is correct, then it also interacts at the proposed site, probably utilizing similar P-450cam electrostatic contacts.     

Single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin

A study of the single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin was performed using the inhibitor metyrapone to trap the cytochrome immediately after release of the product, 5-exo-hydroxycamphor. EPR determinations of the concentrations of reduced putidaredoxin and ferric metyrapone-bound cytochrome at the same time points showed that there is no time lag between the oxidation of reduced putidaredoxin and the appearance of metyrapone-bound cytochrome. This implies that the rate constant for electron transfer is smaller than the rate constant for the later processes involved in product formation and release, lumped into a single step. Taking this restriction into account and doing computer simulation of absorbance versus time curves, previously obtained at various putidaredoxin concentrations using stopped-flow spectrophotometry, allowed bounds to be determined for rate constants of the processes within the reaction. At 4 degrees C in buffer at pH 7.4 with 0.50 M KCl, the rate constant for the bimolecular association of the two enzymes is between 3 and 20/microM.s; the rate constant for dissociation is between 12 and 600/s; the rate constant for electron transfer is between 60 and 100/s; and the rate constant for the later processes is at least 200/s.     

The cytochrome P-450cam binding surface as defined by site-directed mutagenesis and electrostatic modeling

Cytochrome P-450cam cationic surface charges at Lys 344, Arg 72, and Lys 392 have been altered by site-directed mutagenesis techniques. The residues at Lys 344 and Arg 72 were previously suggested as salt bridge contacts in the cytochrome b5-cytochrome P-450cam association complex and implicated in the physiological putidaredoxin-cytochrome P-450cam complex [Stayton, P. S., Poulos, T. L., & Sligar, S. G. (1989) Biochemistry 28, 8201-8205]. Mutations to neutralize the basic charge at Arg 72 (R72Q) and to both neutralize and reverse the charge at Lys 344 (K344Q, K344E) resulted in alteration of NADH oxidation rates in the reconstituted physiological electron-transfer system, which is rate limited by putidaredoxin-cytochrome P-450cam electron transfer. The steady-state Vmax values were apparently unperturbed, suggesting that the observed rate differences were largely attributable to Km effects. The Km values observed for the K344Q (24 microM) and K344E (32 microM) mutants are in the direction expected for neutralization and reversal of a salt bridge charge interaction. A control mutation at a basic surface charge located away from the proposed site of interaction, Lys 392 (K392Q), resulted in overall activities quantitated by NADH oxidation rates that are similar to that of wild-type cytochrome P-450cam. Calculation of the cytochrome P-450cam electrostatic field revealed a patch of positive potential at the modeled cytochrome b5 interaction site lying directly above the nearest proximal approach to the buried heme prosthetic group. These results provide experimental and theoretical evidence for the modeled cytochrome P-450cam binding site implicated in both cytochrome b5 and putidaredoxin association.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observation of the O-O stretching Raman band for cytochrome P-450cam under catalytic conditions

Dioxygen stretching (voo) Raman band was observed for the oxy form of Pseudomonas putida cytochrome P-450 (P-450cam) generated at room temperature under catalytic conditions, that is, in the presence of D-camphor, beta-NADH, putidaredoxin, and putidaredoxin reductase, by using the mixed flow transient Raman apparatus. At the same time the visible absorption spectra were monitored for the transient species. It was found that the voo frequency is little altered by binding of putidaredoxin to P-450cam, although the reduction rate of the oxy form becomes faster. Another intermediate with an oxygen isotope-sensitive band was not found in a time region until 2 s after mixing of the reduced enzyme with oxygen.     

Cloning and nucleotide sequences of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Pseudomonas putida

Pseudomonas putida PpGl, which carries the CAM plasmid encoding enzymes involved in the degradation pathway of D-camphor, can utilize D-camphor as a sole carbon source. Cytochrome P-450cam and related enzymes participate in the early oxidation steps of D-camphor degradation metabolism. We cloned from a HindIII DNA library of PpGl a 2.9 kbp CAM segment which carries the major part of camA gene encoding NADH-putidaredoxin reductase and the entire camB gene encoding putidaredoxin. The 2.9 kbp CAM segment was adjacent to the 4.27 kbp HindIII CAM segment which has been previously cloned (Koga et al. (1986) J. Bacteriol. 166, 1089-1095). Thus, the total 7.17 kbp HindIII CAM directed all the genes responsible for early steps of D-camphor degradation, i.e. 5-exo-hydroxycamphor dehydrogenase (camD gene), cytochrome P-450cam (camC), NADH-putidaredoxin reductase (camA), and putidaredoxin (camB). These cam genes form an operon, camDCAB, and are under negative control by the gene camR located immediately upstream from the camD gene. The total number of amino acids deduced from the nucleotide sequence is 422 for putidaredoxin reductase, and 106 for putidaredoxin.     

Autooxidation and hydroxylation reactions of oxygenated cytochrome P-450cam.

Oxy-ferrous substrate-bound cytochrome P-450cam (mrsO2) autooxidizes in the absence of its specific effector protein, putidaredoxin, without hydroxylating the substrate, camphor. The autooxidation is first order with an activation energy of 17 kcal mol-1 at 25 degrees, pH 7.0. Substrate removal and low pH accelerate the reaction. The product, 5-exo-OH camphor, and a nonhydroxylated pseudosubstrate, norcamphor, stabilize the complex in a manner similar to camphor. Increased oxidation rate of mrsO2 and substrate hydroxylation are induced by putidaredoxin, rebredoxin, cytochrome b5, and the apoproteins of the latter two. Dihydrolipoic acid and other dithiols also replace putidaredoxin as effector molecules, but 1000-fold higher concentrations are required. Effector molecules do not increase the autooxidation rate of mrsO2 unless camphor, norcamphor, or another pseudosubstrate is present. Kinetic evidence is presented showing that an active complex between mrsO2 and effector is a required intermediate in mixed function oxidation.     

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.     

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.     

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.     

Genetic variants in the putidaredoxin-cytochrome P-450cam electron-transfer complex: identification of the residue responsible for redox-state-dependent conformers.

Camphor is hydroxylated in Pseudomonas putida by a three-component system comprised of an oxidase, cytochrome P-450cam, and a two-protein electron-transfer chain, putidaredoxin and putidaredoxin reductase [Tyson et al. (1972) J. Biol. Chem. 274, 5777-5784]. The enzymatic removal of putidaredoxin's C-terminal tryptophan is known to cause a much reduced rate of enzymatic activity in the reconstituted camphor hydroxylase system [Sligar et al. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3906-3910]. To further study the role of tryptophan in the association and/or electron-transfer reactions of putidaredoxin, the gene coding for the iron-sulfur protein was altered so that the tryptophan codon was either deleted or replaced by Phe, Tyr, Asp, Leu, Val, or Lys. Although the initial evaluation of these variant proteins [Davies et al. (1990) J. Am. Chem. Soc. 112, 7396-7398] showed much reduced velocities of electron transfer between P-450cam and the nonaromatic C-terminal proteins, the relative contributions of the binding specificity and intracomplex electron-transfer rates were not addressed. We report here a complete kinetic characterization of these proteins where the dependence of the rate constant on the putidaredoxin concentration was used to determine the intracomplex electron-transfer rate constants and the association energies for all the putidaredoxins in both oxidation states. The sum of forward and reverse intracomplex electron-transfer rate constants varies from 4.90 s-1 for the Lys C-terminal variant to 172 s-1 for the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cysteinyl-tRNA formation: the last puzzle of aminoacyl-tRNA synthesis

A 1H nuclear magnetic resonance study of the complex of cytochrome P450cam-putidaredoxin has been performed. Isocyanide is bound to cytochrome P450cam in order to increase the stability of the protein both in the reduced and the oxidized state. Diprotein complex formation was detected through variation of the heme methyl proton resonances which have been assigned in the two redox states. The electron transfer rate at equilibrium was determinated by magnetization transfer experiments. The observed rate of oxidation of reduced cytochrome P450 by the oxidized putidaredoxin is 27 (+/- 7) per s.     

Steady-state kinetic investigation of cytochrome P450cam: interaction with redox partners and reaction with molecular oxygen

Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that requires two proteins, putidaredoxin reductase (PdR) and putidaredoxin (Pdx), to supply electrons from NADH. This study addresses the mechanism by which electrons are transported from PdR to P450cam through Pdx and used to activate O(2) at the heme of P450cam. It is shown that k(cat)/Km(O2) is independent of the PdR concentration and hyperbolically dependent on Pdx. The phenomenon of saturation of reaction rates with either P450cam or PdR at high ratios of one enzyme to the other is investigated and shown to be consistent with a change in the rate limiting step. Either the reduction of Pdx by PdR (high P450) or the reduction of P450 by Pdx (high PdR) determines the rate. These data support a mechanism where Pdx acts as a shuttle for transport of electrons from PdR to P450cam, effectively ruling out the formation of a kinetically significant PdR/Pdx/P450cam complex.     

Combined QM/MM calculations of active-site vibrations in binding process of P450cam to putidaredoxin

Combined QM/MM calculations of the active-site of cytochrome P450cam have been performed before and after the binding of P450cam to putidaredoxin. The calculations were carried out for both a 5-coordinated and a 6-coordinated active-site of cytochrome P450cam, with either a water molecule or a carbon monoxide molecule as a 6th distal ligand. An experimentally observed increase in the Fe-S stretching frequency that occurs after cytochrome P450cam binds to putidaredoxin, has been reproduced in our study. Experimentally observed changes in the Fe-C and C-O vibration frequencies that occur after binding of both proteins, have also been reproduced in our study. The computed increase of the Fe-S and Fe-C stretching frequencies is correlated with a corresponding decrease of the Fe-S and Fe-C interatomic distances. According to our calculations, for the active-site with carbon monoxide in the triplet electronic state, the binding process increases the spin densities on the iron and sulfur atoms, which changes the Fe-C and C-O stretching frequencies in opposite directions, in agreement with experimental data.     

Determination of cytochrome b5 association reactions. Characterization of metmyoglobin and cytochrome P-450cam binding to genetically engineered cytochromeb5

Genetically engineered cytochrome b5 has been used to quantitative binding interactions of this protein with cytochrome P-450cam and sperm whale metmyoglobin by static fluorescence titration. Two cytochrome b5 mutants were constructed by cassette mutagenesis to replace a surface threonine residue with cysteine at two crystallographically defined positions, 65 and 8, located 11 and 21 A, respectively, from the nearest heme edge. The T65C and T8C mutant proteins were labeled with the sulfhydryl selective fluorescent reagent, acrylodan, which provided a spectral probe for monitoring protein-protein association. The fluorescence emission spectra of the acrylodan-labeled T65C mutant exhibited an ionic strength-dependent, blue-shifted fluorescence enhancement upon binding met-myoglobin, cytochrome c, and cytochrome P-450cam, whereas the acrylodan-labeled T8C mutant fluorescence emission remained unchanged during all titrations. Dissociation constants of 1.3, 0.6, and 0.5 microM, pH 7.15, were measured for metmyoglobin, cytochrome P-450cam, and cytochrome c, respectively. A similar averaged binding surface for cytochrome P-450cam and cytochrome c is suggested by their closely related degree of fluorescence enhancement, degree of emission blue shift, and binding free energies. Myoglobin binds less tightly, enhances fluorescence to a greater extent, and exhibits a larger blue shift in acrylodan emission spectra suggesting a different averaged binding orientation relative to the acrylodan probe.     

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.     

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.     

Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.     

Apoprotein formation and heme reconstitution of cytochrome P-450cam

Apoprotein suitable for heme reconstitution has been prepared by an acid/butanone extraction of cytochrome P-450cam at pH 2.5. Absorption spectra of apo-P-450cam indicate less than 2% residual holoenzyme. Four tryptophan residues per molecule were estimated from the aromatic absorbance region of denatured apoprotein. Heme-reconstituted holoprotein was purified in 30% yield to a specific activity equivalent to the native enzyme. Absorption and EPR spectra of 57Fe- and 54Fe-heme-enriched P-450cam reveal complete restoration of the native active site.