Calibration of ring-current effects in proteins and nucleic acids

Case, David A.

doi:10.1007/bf00197633

Cited by 116 publications

(136 citation statements)

References 26 publications

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“…The NMR work shows that the resonances from the 5-exo and 9-methyl protons of D-camphor and one of the ␤-protons of Cys-357 shift upfield by 1.28, 0.20, and 0.12 ppm, respectively, upon Pdx binding. On the basis of these spectral shifts and the JohnsonBovey model for ring current shift (35,36), such high field shifts indicate that these protons move closer to the heme plane and/or the heme normal passing through the iron atom (z axis) by ϳ0.5, ϳ0.15, and ϳ0.1 Å, respectively. This is qualitatively consistent with the CO-L358P crystal structure because both the C-5 and C-9 atoms of the substrate are closer to the z axis compared with that of the CO-WT complex (Table II and Figs. 3 and 5) by ϳ0.1 and 0.3 Å, respectively.…”

Section: Resultsmentioning

confidence: 91%

Crystal Structure of the Cytochrome P450cam Mutant That Exhibits the Same Spectral Perturbations Induced by Putidaredoxin Binding

Nagano

Tosha

Ishimori

et al. 2004

Journal of Biological Chemistry

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The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdxbinding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with nonphysiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor.Cytochrome P450cam (P450cam) 1 (1, 2) catalyzes the regioand stereo-specific hydroxylation of D-camphor. To activate molecular oxygen, P450cam requires two protons and two electrons. Protons are provided from bulk water through a proton shuttle system that includes Asp-251, Thr-252, and water molecules (3-6). Electrons are provided by an iron-sulfur protein, putidaredoxin (Pdx). In the electron transfer steps, P450cam forms a complex with reduced Pdx, and the P450cam active site is perturbed as indicated by various spectroscopic studies including NMR (7, 8), EPR (9, 10), resonance Raman (11-13), and infrared (14, 15). Because nonphysiological redox proteins such as ferredoxin and adrenodoxin cannot support the monooxygenation reaction or induce the conformational changes (7, 16), the Pdxinduced changes in P450cam are thought to be important for the "effector" function of Pdx in addition to its reductase activity. In support of this view, a recent infrared and mutational study (15) has shown that Pdx-induced conformational changes promote the camphor monooxygenation reaction.Recent NMR studies found that the CO complex of the L358P mutant has very similar spectroscopic property to the WT P450cam-CO in a complex with Pdx (8,17). This suggests that the L358P mutant is a good model for Pdx-bound P450cam. However, our knowledge on the conformational change from the NMR studies is limited to only two residues, the axial cysteine ligand (Cys-357) and Thr-252 and the substrate. To overcome this limitation and to complement the NMR work, we have solved the crystal structures of the ferrous and ferrous-CO complex of the L358P mutant. EXPERIMENTAL PROCEDURESProtein Purification and Crystallization-WT P450cam and the L358P 2 mutant were overexpressed in Escherichia coli and purified as described elsewhere (18). The CO-bound P450cam structure was first solved by Raag and Poulos (19) by using the initial P2 1 2 1 2 1 crystal form used to solve the P450cam structure. For a more accurate...

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Section: Resultsmentioning

confidence: 91%

Crystal Structure of the Cytochrome P450cam Mutant That Exhibits the Same Spectral Perturbations Induced by Putidaredoxin Binding

Nagano

Tosha

Ishimori

et al. 2004

Journal of Biological Chemistry

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“…Chemical shifts were calculated from the coordinates by using the model in the program SHIFTS with the parameters of Ö sapay and Case (30). The application of an alternative parameter set using larger ring current intensity factors based on quantum chemical calculations (31) led to a significant overestimation of the ring current contributions to the chemical shifts as judged by comparison to the experimental results.…”

Section: Methodsmentioning

confidence: 99%

The structures of the active center in dark-adapted bacteriorhodopsin by solution-state NMR spectroscopy

Patzelt

Simon

terLaak

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The two forms of bacteriorhodopsin present in the dark-adapted state, containing either all-trans or 13-cis,15-syn retinal, were examined by using solution state NMR, and their structures were determined. Comparison of the all-trans and the 13-cis,15-syn forms shows a shift in position of about 0.25 Å within the pocket of the protein.Comparing this to the 13-cis,15-anti chromophore of the catalytic cycle M-intermediate structure, the 13-cis,15-syn form demonstrates a less pronounced up-tilt of the retinal C12OC14 region, while leaving W182 and T178 essentially unchanged. The NOH dipole of the Schiff base orients toward the extracellular side in both forms, however, it reorients toward the intracellular side in the 13-cis,15-anti configuration to form the catalytic M-intermediate. Thus, the change of the NOH dipole is considered primarily responsible for energy storage, conformation changes of the protein, and the deprotonation of the Schiff base. The structural similarity of the all-trans and 13-cis,15-syn forms is taken as strong evidence for the ion dipole dragging model by which proton (hydroxide ion) translocation follows the change of the dipole.A number of important biological processes, including the reception of hormon-or light-encoded signals, depend on the function of hepta-helical transmembrane proteins (1, 2). Certain members of this family, like the visual pigment rhodopsin and the archaeal proton pump bacteriorhodopsin (BR), contain a light reactive prosthetic retinal group that is attached via a protonated Schiff base linkage (ϪC ϭ NHϩ) to the -amino group of a lysine residue in the seventh helix. Their light sensitivity is based on photoisomerization of this retinal moiety. The specific amino acid composition in the active center determines the wavelength of chromophore absorption, the energy barriers for bond rotation in the retinylidene moiety, and the regioselectivity of isomerization. Pioneering structural investigations were done with electron microscopy (3). Recently, high-resolution three-dimensional structures of BR (4-13) (initial state and M-, K-intermediates), the related halorhodopsin (14), and rhodopsin (15) have been made available.Bacteriorhodopsin converts light energy into that of a proton gradient that is subsequently used by the transmembrane protein ATP-synthase to produce chemical energy in the form of ATP. At the beginning of the photocycle, the catalytically active form of BR absorbs a photon by the all-trans,15-anti retinal cofactor that is linked to K216 forming a Schiff base. During the photoexcitation, the retinal undergoes a rearrangement of the electronic structure of its extended conjugated system, resulting in the trans 3 cis isomerization to a 13-cis,15-anti form, and causing reduced proton affinity on the charged Schiff base. The M intermediate is formed by the transfer of a proton from the Schiff base to D85 in the extracellular half-channel. Reprotonation of the Schiff base is mediated by D96 in the cytoplasmic half-channel of the protein in response to the larg...

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“…Because the contribution of the ring current shifts mainly depend on the position of the proton relative to the heme plane (29,30), the ring current-shifted signals for the L358P mutant provide more detailed information on the local heme environment of the L358P mutant. By using the Johnson Bovey model (29), a useful tool for calculating the ring current contribution, the chemical shifts in the mutant indicate that the ␤-proton of the axial Cys and the 9-methyl group and 5-exo proton of Dcamphor approach closer to the heme iron by ϳ0.15, 0.1, and 0.2-0.3 Å, respectively, upon the mutation.…”

Section: Heme Environment Of the Ferrous-co Form Of L358p-up-mentioning

confidence: 99%

L358P Mutation on Cytochrome P450cam Simulates Structural Changes upon Putidaredoxin Binding

Tosha¹,

Shiro²,

Ishimori

et al. 2004

Journal of Biological Chemistry

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Calibration of ring-current effects in proteins and nucleic acids

Cited by 116 publications

References 26 publications

Crystal Structure of the Cytochrome P450cam Mutant That Exhibits the Same Spectral Perturbations Induced by Putidaredoxin Binding

Crystal Structure of the Cytochrome P450cam Mutant That Exhibits the Same Spectral Perturbations Induced by Putidaredoxin Binding

The structures of the active center in dark-adapted bacteriorhodopsin by solution-state NMR spectroscopy

L358P Mutation on Cytochrome P450cam Simulates Structural Changes upon Putidaredoxin Binding

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