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PubMed1991

A hypervariable region of P450IIC5 confers progesterone 21-hydroxylase activity to P450IIC1.

Cytochrome P450IIC5 is a hepatic progesterone 21-hydroxylase while the 95% identical P450IIC4 has a greater than 10-fold higher Km for progesterone 21-hydroxylation and the 74% identical P450IIC1 does not hydroxylate progesterone at detectable rates. Previous work demonstrated that the apparent Km of P450IIC4 for progesterone 21-hydroxylation can be markedly improved by replacing a valine at position 113 with an alanine which is present at this position in P450IIC5. In the present studies, a single point mutation in cytochrome P450IIC1 that changed valine at position 113 to alanine conferred progesterone 21-hydroxylase activity to this enzyme. Although the catalytic activity was less than that of P450IIC5, these results indicate the residue 113 plays a critical role in the determination of the substrate/product selectivity in subfamily IIC P450s. By alignment with the sequence of P450cam, the segment of the polypeptide, residues 95-123, containing residue 113 corresponds to a substrate-contacting loop in the bacterial enzyme. The region containing residue 113, which is highly variable among family II P450s, may also be a substrate-contacting loop in the mammalian cytochromes P450. The exchange of this hypervariable region of cytochrome P450IIC1, residues 95-123, with that of P450IIC5 enhanced the 21-hydroxylase activity of the cells transfected with this chimera to levels similar to those of cells transfected with the plasmid encoding P450IIC5. Kinetic analysis of microsomes isolated from the transfected cells showed that the apparent Km for progesterone 21-hydroxylation of the chimera was indistinguishable from that of P450IIC5.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2001

Resonance Raman and EPR investigations of the D251N oxycytochrome P450cam/putidaredoxin complex.

We have performed resonance Raman and electron paramagnetic resonance (EPR) studies on the dioxygen bound state of the D251N mutant of cytochrome P450cam (oxy-P450cam) and its complex with reduced putidaredoxin (Pd). The D251N oxy-P450cam/Pd complex has a perturbed proton delivery mechanism and shows a significantly red-shifted UV-visible spectrum as observed in Benson et al. [Benson, D. E., Suslick, K. S., and Sligar, S. G. (1997) Biochemistry 36, 5104-5107]. The red shift has been interpreted to indicate a major perturbation of the electronic structure of the oxy-heme complex. However, we find no evidence that electron transfer has occurred from Pd to the heme active site of D251N oxy-P450cam. This suggests that both electron and proton transfer are perturbed by the D251N mutation and that these processes may be coupled. Three oxygen isotope sensitive Raman features are identified in the Pd complex, and occur at 1137, 536, and 399 cm(-1). These values are not significantly different from those for WT or D251N oxy-P450cam. However, a careful examination of the oxygen stretching feature near 1137 cm(-1) reveals the presence of three peaks at 1131, 1138, and 1146 cm(-1), which we attribute to the presence of conformational substates in oxy-P450cam. A significant change in the conformational substate population is observed for the D251N oxy-P450cam when the Pd complex is formed. We suggest that the conformational population redistribution of oxy-P450cam, along with the red-shifted electronic spectra, reflects a structural equilibrium of the oxy-heme that is perturbed upon Pd binding. We propose that this structural perturbation is connected to the effector function of Pd and may involve changes in the electron donation properties of the thiolate ligand.

Amino Acid Substitution↗
PubMed1984

NADH- and oxygen-dependent multiple turnovers of cytochrome P-450-CAM without putidaredoxin and putidaredoxin reductase.

Phenazine methosulfate (PMS) has been successfully used to mediate electron transfer from NADH to cytochrome P-450-CAM in the absence of putidaredoxin and putidaredoxin reductase under aerobic conditions. Identification and quantitation of exo-5- hydroxycamphor , the only product, has been accomplished by gas chromatography. In the absence of cytochrome P-450-CAM, or when other heme proteins (hemoglobin, myoglobin, horseradish peroxidase) are substituted for P-450-CAM, no exo-5- hydroxycamphor is detected. Product formation is not inhibited by the addition of catalase, superoxide dismutase, or hydroxyl radical scavengers; however, significant inhibition is observed with carbon monoxide and metyrapone, known inhibitors of the fully reconstituted P-450 system. Addition of 2,3-dimercaptopropanol to the NADH/PMS/P-450 system leads to a 4-fold increase in product formation; when putidaredoxin is added (without dimercaptopropanol), a 20-fold increase in product formation is observed. Constant bubbling with oxygen results in a further increase in the amount of product (150-fold increase overall). Our results show that PMS can substitute for the electron-transfer proteins putidaredoxin and putidaredoxin reductase in the transfer of electrons from NADH to P-450-CAM, resulting in multiple turnovers. Molecular oxygen dependent multiple turnovers of cytochrome P-450 have not been previously observed without the fully reconstituted, three-protein system.

Camphor↗
PubMed1989

Secondary structure prediction of 52 membrane-bound cytochromes P450 shows a strong structural similarity to P450cam.

The secondary structure of 52 aligned cytochrome P450 sequences, all of which are membrane bound, is predicted and collectively compared with the crystal structure of the soluble cytochrome P450cam. Ten of 13 helical regions, 6 of 7 beta-pair regions, and beta-structure corresponding to a known beta-bulge near the active site of P450cam are predicted to exist in the membrane-bound P450s. Three turns associated with beta-structure in the soluble enzyme are also predicted for the membrane-bound forms. A strong structural similarity is evident between membrane P450s and the soluble P450cam. Consequently, a multitransmembrane structure involving much of P450 seems highly unlikely. A structure with two N-terminal transmembrane segments is compatible with these observations.

Animals↗
PubMed1989

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.

Binding, Competitive↗
PubMed1990

Tyrosine-96 as a natural spectroscopic probe of the cytochrome P-450cam active site.

The previously described correlation between the ferric spin equilibrium of cytochrome P-450cam and the environmental polarity of tyrosine residues (Fisher et al., 1986) has been further examined with the use of site-directed mutagenesis and active-site affinity reagents. Whereas the wild-type demonstrates an increase in environmental polarity of approximately one tyrosine residue, the mutant protein Y96F, in which Tyr-96 has been changed to Phe-96, demonstrates a lack of spin-state-dependent change in the second-derivative ultraviolet absorption spectrum. This suggests that the active-site Tyr-96 serves as a ultraviolet spectroscopic probe which can be utilized to determine the relative degree of water access to the active site for various substrate/protein complexes. The affinity reagent isobornyl mercaptan has been used to demonstrate the utility of this probe in determining the active-site polarity when substrate analogues are bound at the active site. In addition, the sensitivity of Tyr-96 to environmental polarity has been used to demonstrate that the product/enzyme complex, formed with 5-exo-hydroxycamphor, may be associated with increased water access to the heme iron. This may provide a means for turning off electron transfer when the product, instead of the substrate, is bound at the active site.

Binding Sites↗
PubMed1990

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)

Binding Sites↗
PubMed2001

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.

Camphor 5-Monooxygenase↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2003

Crystallographic studies on the complex behavior of nicotine binding to P450cam (CYP101).

Crystallographic and spectroscopic studies have been undertaken to characterize the binding behavior of the non-native substrate nicotine in the active site of the monooxygenase hemoprotein cytochrome P450cam. Despite the existence of a theoretical model that is consistent with the observed distribution of monooxygenation products, the crystal structure of the complex indicates that the primary binding mode of nicotine is unproductive. The structure is confirmed by spectral data that indicate direct coordination of substrate pyridine nitrogen with the heme iron. This would be the proper structure for evaluating binding affinity and inhibition. Reduction of the heme from Fe(III) to Fe(II) and introduction of carbon monoxide into crystals of the nicotine-P450cam complex, to simulate molecular oxygen binding, produces reorientation of the nicotine. This orientation is the appropriate one for predicting regioselectivity and the kinetic features of substrate oxidation. While it is not clear that such complicated behavior will be exhibited for other enzyme-substrate interactions, it is clear that a single crystal structure for a given substrate-enzyme interaction may not provide a good description of the binding mode responsible for product formation.

Binding Sites↗
PubMed2004

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.

Bacterial Proteins↗
PubMed2004

Structural stability and dynamics of hydrogenated and perdeuterated cytochrome P450cam (CYP101).

Perdeuterated and hydrogenated cytochrome P450cam (P450cam), from Pseudomonas putida, has been characterized concerning thermal stability and structural dynamics. For the first time, Fourier transform infrared (FTIR) spectroscopy was used to characterize a perdeuterated protein. The secondary structure compositions were determined from the fitted amide I' spectral region, giving band populations at 10 degrees C for the perdeuterated protein of 22% between 1605 and 1624 cm(-1) (beta-sheets), 47% between 1633 and 1650 cm(-1) (alpha-helix (29%) plus unordered/3(10)-helix (18%)), and 28% between 1657 and 1677 cm(-1) (turns) and for the hydrogenated protein of 22% between 1610 and 1635 cm(-1) (beta-sheets), 52% between 1640 and 1658 cm(-1) (alpha-helix (41%) plus unordered/3(10)-helix (11%)), and 24% between 1665 and 1680 cm(-1) (turns). Thermal unfolding experiments revealed that perdeuterated P450cam was less stable than the hydrogenated protein. The midpoint transition temperatures were 60.8 and 64.4 degrees C for the perdeuterated and hydrogenated P450cam, respectively. Step-scan time-resolved FTIR was applied to the P450cam-CO complex to study the ligand-rebinding process after flash photolysis. Rebinding of the ligand occurred with the same kinetics and rate constants k(on), 8.9 x 10(4) and 8.3 x 10(4) M(-1) s(-1) for the perdeuterated and hydrogenated P450cam, respectively.Perdeuterated P450cam was expressed for a neutron crystallographic study to determine the specific hydration states and hydrogen-bonding networks at the active site. The analyses presented here show that perdeuterated P450cam is structurally similar to its hydrogenated counterpart, despite its reduced thermal stability, suggesting that information obtained from the neutron structure will be representative of the normal hydrogenated P450cam.

Binding Sites↗
PubMed2006

Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes.

Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.

Alanine↗
PubMed2006

Role of threonine 101 on the stability of the heme active site of cytochrome P450cam: multiwavelength circular dichroism studies.

The role of the threonine 101 residue that resides close to the heme propionic acid side chain of cytochrome P450cam on the conformational properties of the active site of the enzyme has been investigated by circular dichroism (CD) spectroscopy. Site-specific mutation of the threonine by valine has been carried out that does not affect the size of the residue but significantly alters the hydropathy index. The T101V mutant of cytochrome P450cam showed distinct differences in the CD spectra near the heme region, indicating a subtle effect of the mutation on the properties of the heme active site. Thermal stabilities of the mutant and wild-type enzyme have been studied by temperature dependence of the ellipticity (intensity of the CD band) in the far-UV region for the secondary structure and at different wavelengths in the visible region that arise from the heme moiety for the tertiary structure around the prosthetic group. The thermal unfolding data from variations of the CD intensity at different wavelengths were analyzed using a generalized multistep unfolding model, and two distinct equilibrium intermediate conformational states of the enzyme were identified. The mutation of the T101 residue by valine was found to decrease the thermal stability of both the intermediates in the presence of the substrate. On the other hand, this mutation had no apparent effect on the thermal stability of the enzyme in the absence of the substrate. These results suggested that the threonine residue stabilizes the protein cavity around the heme center in the case of the substrate-bound species, possibly by hydrogen bonding with one of the propionate side chains of the heme moiety. Such hydrogen bonding of the heme propionate with threonine is absent in the substrate-free form of the enzyme.

Amino Acid Sequence↗
PubMed1996

Role of the polarity of the heme environment for the CO stretch modes in cytochrome P-450cam-CO.

The CO stretch mode of various substrate complexes of cytochrome P-450cam-CO was measured using FT infrared spectroscopy. At room temperature most of the complexes show a single, but often asymmetric infrared band. The representative wavenumber of this band for the various complexes increases when the high-spin content, induced by the substrates in the oxidized protein, decreases. Additionally, the increase of the CO stretch wavenumber (1939 to 1956 cm-1) correlates with the decrease of the Soret band wavenumber (22440 to 22373 cm-1). It is suggested that the polarity of the heme pocket is modulated by the substrates due to changed accessibility of the heme environment for water molecules. The increased water content compensates positive electrostatic potentials near the CO ligand, which results in loosening the contact of CO to the I helix.

Camphor 5-Monooxygenase↗
PubMed1997

Reduced oxy intermediate observed in D251N cytochrome P450cam.

Cytochrome P450s are ubiquitous heme proteins responsible for various oxidative metabolic processes. The overall rate-determining step in the catalytic cycle of native cytochrome P450cam is the reduction of the dioxygen complex, which has made detection of catalytic intermediates after this reduction impossible. However, for the site-specific mutant D251N cytochrome P450cam (which affects proton transfer near the catalytic center), the overall rate-determining step occurs after the reduction of oxy-P450. As a consequence, we have observed in the UV-visible spectrum during catalytic turnover a new intermediate that is one electron reduced from oxy-P450 with an intact dioxygen bond.

Camphor 5-Monooxygenase↗