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.     

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)     

Single turnover studies with oxy-cytochrome P-450cam

The catalytic step of bacterial cytochrome P-450cam, i.e., the step of the reaction cycle in which the product 5-exo-hydroxycamphor is formed and released by the enzyme, has been studied by stopped-flow spectrophotometry. Our approach has been to observe a single-turnover reaction between reduced putidaredoxin and oxygenated camphor-bound cytochrome P-450cam. Multiple turnovers are prevented by using the inhibitor metyrapone to trap the cytochrome after product release, which prevents binding of another camphor molecule. The time course of the reaction has been measured at several wavelengths and has been found to be biphasic. The relatively slow second phase of the reaction is the reduction of ferric, metyrapone-bound cytochrome P-450cam. The first phase coincides with the formation of product stoichiometrically with cytochrome P-450cam, as measured by gas chromatography. A detailed kinetic study of the first phase reveals a hyperbolic dependence of initial rate upon putidaredoxin concentration at a fixed, limiting concentration of cytochrome P-450cam. The Vmax is 53 microM per second per microM cytochrome, and the Km for putidaredoxin is 33 microM. The hyperbolic relationship between initial rate and putidaredoxin concentration supports a model in which the cytochrome rapidly binds putidaredoxin, then undergoes one or more slower intracomplex steps.     

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.     

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.     

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.     

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.     

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.     

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.     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.     

Primary and secondary structural patterns in eukaryotic cytochrome P-450 families correspond to structures of the helix-rich domain of Pseudomonas putida cytochrome P-450cam. Indications for a similar overall topology

An extensive sequence analysis of the eukaryotic cytochrome P-450 (P-450) protein families was conducted with a view to identifying conserved regions that might be related to secondary structural features in the Pseudomonas putida camphor hydroxylase (P-450cam). All sequences available on-line were collected, classified and aligned within families. Distinctively different sequences were chosen from each of seven eukaryotic families, and an unbiased multi-alignment was constructed. Profile patterns of the most conserved regions were generated and screened against the sequence of P-450cam, the structure of which has been elucidated by X-ray crystallography. While some of these profiles did not map on the P-450cam sequence, the structurally most important helices were clearly identified and the correlations were found to be statistically significant. Our analysis suggests that the helix-rich domain with the cysteine pocket and the oxygen-binding site is conserved in all P-450 forms. Helices I and L from P-450cam can be easily identified in all eukaryotic P-450 forms. Other helices which seem to exist in all P-450 forms include helices C, D, G and J. K. In the helix-poor domain of P-450cam, only structures b3/b4 seem to have been conserved. The obvious sequence conservation throughout the helix-rich domain of the P-450cam protein might be expected for a molecular class whose overall topology is preserved. Additional support for the conservation of structure between eukaryotic cytochromes P-450 and P-450cam comes from secondary structure prediction of the eukaryotic sequences.     

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)     

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.     

Conformational changes of cytochromes P-450cam and P-450lin induced by high pressure

Absorbance and fluorescence spectra of bacterial cytochrome P-450cam and cytochrome P-450lin have been studied as a function of pressure. These pressure-induced spectral perturbations fall into two categories, which are interpreted as resulting from denaturation domains and are discussed in terms of protein structural dynamics. The results presented herein support a view that these two bacterial cytochromes have large structural differences and suggest a picture in which the gellike cortex of each protein may play an essential role in stability and function.     

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.     

Putidaredoxin-cytochrome P450cam interaction

Cytochrome P450cam (P450cam) catalyzes the monooxygenation of D-camphor. During the enzymatic reaction, oxyferrous, D-camphor-bound P450cam forms a binary complex with reduced putidaredoxin as an obligatory reaction intermediate. We have found that reduced putidaredoxin undergoes EPR-detectable conformational changes upon formation of the intermediate complex and also upon formation of a binary complex with CO- or NO-ferrous, D-camphor-bound P450cam. The structural changes in putidaredoxin are almost identical irrespective of the ligand bound to P450cam, and distinct from and significantly larger than those induced by unliganded ferrous P450cam. The binary complex formation also induce conformational alterations in the CO- and NO-ferrous, D-camphor-bound P450cam, thereby evoking simultaneous changes in the structure of the two proteins. A molecular basis and roles of such structural changes in the D-camphor monooxygenation are discussed.     

Electrochemical analysis of microsomal cytochromes]

The electrochemical behaviour of the electron transfer proteins -- cytochromes b5, c and P-450 was studied by classical polarography and electrolysis with spectrophotometric monitoring. It was shown electrons are directly transferred from the electrode to oxidized cytochromes c and b5. Cytochrome P-450 is not reduced on the electrodes. However, the reduced inactivated from of cytochrome P-420 was detected at high potential values (-1,5 v).     

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 organization and localization of cytochromes P450 in the membrane

The secondary structure prediction of 19 microsomal cytochrome P-450s from two different families was made based on their amino acid sequences. It was shown that there is a structural similarity between the heme-binding sites of these enzymes and the bacterial P-450cam. An average predicted secondary structure of cytochrome P-450 proteins with 70% accuracy contains about 46% alpha-helices, 12% beta-strands, 9% beta-turns and 33% random coil. In the region of the 35-120 residues in microsomal P-450s two adjacent beta alpha beta-units (the Rossmann domain) were recognized, which may interact with the NADPH-cytochrome P-450 reductase. Using the procedure of identification of hydrophobic and membrane-associated alpha-helical segments of 23 cytochromes, only one N-terminal transmembrane anchor was predicted. Also the heme-binding site perhaps includes surface-bound helix. A model of vertebrate microsomal P-450s is proposed. That is an amphypathic membrane protein located on the cytoplasmic face of the endoplasmic reticulum, their active center lies out/on the bilayer border.