Camphor-bound Cytochrome P450cam Research Articles

Derivation of Mean-Square Displacements for Protein Dynamics from Elastic Incoherent Neutron Scattering

The derivation of mean-square displacements from elastic incoherent neutron scattering (EINS) of proteins is examined, with the aid of experiments on camphor-bound cytochrome P450cam and complementary molecular dynamics simulations. It is shown that a q(4) correction to the elastic incoherent structure factor (where q is the scattering vector) can be simply used to reliably estimate from the experiment both the average mean-square atomic displacement, <Δr(2)> of the nonexchanged hydrogen atoms in the protein and its variance, σ(2). The molecular dynamics simulation results are in broad agreement with the experimentally derived <Δr(2)> and σ(2) derived from EINS on instruments at two different energy resolutions, corresponding to dynamics on the ∼100 ps and ∼1 ns time scales. Significant dynamical heterogeneity is found to arise from methyl-group rotations. The easy-to-apply q(4) correction extends the information extracted from elastic incoherent neutron scattering experiments and should be of wide applicability.

Temperature Dependent Dynamics of Cytochrome P450cam from Elastic Incoherent Neutron Scattering

The derivation of mean-square displacements from elastic incoherent neutron scattering (EINS) of proteins is examined, with the aid of experiments on camphor-bound cytochrome P450cam and complementary molecular dynamics simulations. It is shown that a q4 correction to the elastic incoherent structure factor (where q is the scattering vector) can be simply used to reliably estimate from the experiment both the average mean-square atomic displacement (MSD) of the non-exchanged hydrogen atoms in the protein and its variance. The molecular dynamics simulation results are in broad agreement with the experimentally-derived MSD and its variance derived from EINS on instruments at two different energy resolutions, corresponding to dynamics on the ∼100 ps and ∼1 ns timescales. Significant dynamical heterogeneity is found to arise from methyl-group rotations. The easy-to-apply q4 correction extends the information extracted from elastic incoherent neutron scattering experiments and should be of wide applicability.

Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

Resonance Raman spectra are reported for substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD(3) groups at the 1, 3, 5, and 8-positions (d12-protoheme) or deuterated methine carbons (d4-protoheme). In agreement with previous studies of this and similar enzymes, substrate binding induces changes in the high frequency and low frequency spectral regions, with the most dramatic effect in the low frequency region being activation of a new mode near 367 cm(-1). This substrate-activated mode had been previously assigned as a second "propionate bending" mode (Chen et al., Biochemistry, 2004, 43, 1798-1808), arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm(-1). In this work, this newly activated mode is observed to shift by 8 cm(-1) to lower frequency in the d12-protoheme reconstituted enzyme (i.e., the same shift as that observed for the higher frequency "propionate bending" mode) and is therefore consistent with the suggested assignment. However, the newly acquired data for the d4-protoheme substituted analogue also support an earlier alternate suggestion (Deng et al., Biochemistry, 1999, 38, 13699-13706) that substrate binding activates several heme out-of-plane modes, one of which (gamma(6)) is accidentally degenerate with the 367 cm(-1) propionate bending mode. Finally, the study of the enzyme reconstituted with the protoheme-d4, which shifts the macrocycle nu(10) mode, has now allowed a definitive identification of the vinyl C=C stretching modes.

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 −373 mV vs Ag/AgCl 1 M 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.

The CO Stretching Mode Infrared Spectrum of Substrate‐Free Cytochrome P ‐450cam—CO

The effect of pH, glycerol, temperature, and pressure on the carbon monoxide (CO) stretch mode of substrate-free cytochrome P-450cam (CYP101) was studied. Complex spectra of overlapping bands have been observed. CO stretch bands centered at about 1911-1918 cm-1 (band I), 1927-1931 cm-1 (band II), 1940-1942 cm-1 (band III), 1950-1953 cm-1 (band IV), 1960-1963 cm-1 (band V) and 1966-1973 cm-1 (band VI) are obtained from the fitting analyses independently of the lineshape model used. Only two or three bands are dominant in each spectrum. Compared to bands I, II and III, the bands IV and V are assigned to correspond to a weaker polar contact between the CO ligand and a polar group in the heme pocket (probably Thr252) because of the opposite effect of glycerol (osmotic pressure) and hydrostatic pressure on the intensity and frequency of these bands. The different CO stretch bands are interpreted as indicating conformational substates of the protein. It is suggested that water in the heme pocket plays an important role for the substate equilibrium. This substate equilibrium freezes in at the glass transition temperature, Tg, of the protein/solvent mixture. For the temperature region above Tg the thermodynamic parameters and volumes for the substates have been determined by a global fit analysis of the temperature- and pressure-dependent populations and they are compared to the respective values for camphor-bound cytochrome P-450cam.

The carbon monoxide stretching modes in camphor-bound cytochrome P-450cam. The effect of solvent conditions, temperature, and pressure.

The effect of pH, monovalent cations, glycerol, temperature, and pressure on the carbonmonoxy (CO) stretching mode of camphor-bound cytochrome P-450cam (CYP 101) was studied. Two effects, band overlap and frequency shift, have been observed. The CO stretch infrared band located at about 1940 cm-1 is asymmetric because of the overlap of three bands at about 1931 cm-1, 1939 cm-1, and 1942 cm-1 with strongly different populations. Reducing the temperature or increasing the pressure leads to splitting the band or switching the asymmetry from the lower energy side to the higher energy side of the infrared band. The overlap of several CO stretch bands indicates conformational substates within the heme pocket. A frequency shift of the predominantly populated band is observed by changing all the parameters mentioned. The pH-induced frequency shift follows an S-shape with the pK at 6.2, which matches the pK observed for the pH-induced high-spin/low-spin transition. Conformational changes on the proximal heme side are suggested to be the origin. Monovalent cations at saturating concentration induce a small frequency shift depending on the ion radius. The potassium ion is the one that induces a CO stretch frequency with the highest wave-number while sodium and lithium (smaller radii) and rubidium and caesium ion (larger radii) have diminished values, which is supporting evidence for the special function of the potassium ion within the structure. Glycerol and hydrostatic pressure induce a red shift of the CO stretching frequency. Forced contact of the polar hydroxyl group of Thr252 of the I helix induced by pressure and indirectly by glycerol is suggested to change the CO dipole moment, reflecting in the decreased CO stretching frequency.

The proton activity at cryogenic temperatures — A possible influence on the spin state of the heme iron of cytochrome P-450 cam in supercooled buffered solutions #

The electronic absorption spectra for camphor-bound cytochrome P-450 cam have been analysed in the temperature range between 78 K and 298 K. The well-known high-spin/low-spin equilibrium has been detected between 298 K and 220 K. Depending on the cooling rate, below 220 K a new species was found in the absorption spectra. In contrast, the electronic absorption spectra for camphor-free cytochrome P-450 cam between 78 K and 295 K show no significant spectral changes. The conversion between the spin states of camphor-bound cytochrome P-450 cam and the appearance of the new species do not correspond to the temperature-induced change in the p a H value of the aqueous glycerol mixture containing phosphate or cacodylate buffer (p a H 7.0). For this study a spectroscopic procedure for the determination of the temperature dependence of the p a H value of the solvent for the range 78–295 K is presented using dyes as pH-indicators. It is shown that the state of the acid-base equilibrium frozen in is strongly dependent on the cooling rate of the mixture.

Analysis of Active Site Motions from a 175 picosecond Molecular Dynamics Simulation of Camphor-bound Cytochrome P450cam

The structure and internal motions of the active site residues of camphor-bound cytochrome P450cam have been evaluated on the basis of a 175 psec molecular dynamics simulation. The active site residues generally show very small deviations away from their starting crystal positions. These residues also generally show much smaller fluctuations than for the enzyme as a whole. Phe 87 is dynamically very unusual and is suggested to play a role in substrate movement into and/or out of the active site. The average distance between the heme iron and atoms C5, C6, and C3 of camphor is 5.3, 6.0, and 7.0 A, respectively. This trend is consistent with the experimentally observed stereospecificity of the hydroxylation reaction. On the basis of distance and angle criteria, both 5-exo and 5-endo hydrogen abstraction are predicted to occur during the hydroxylation reaction; although the 5-exo pathway is expected to be 3-fold more likely.

Single turnover studies with oxy-cytochrome P-450 cam

The catalytic step of bacterial cytochrome P-450 cam, 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-450 cam. 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-450 cam. The first phase coincides with the formation of product stoichiometrically with cytochrome P-450 cam, 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-450 cam. The V max is 53 μ m per second per μ m cytochrome, and the K m for putidaredoxin is 33 μ m. 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.