Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam

Schulze, Heike; Hoa, Gaston Hui Bon; Jung, Christiane

doi:10.1016/s0167-4838(96)00192-6

Cited by 28 publications

(47 citation statements)

References 38 publications

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“…For WT, the affinity for the different substrates is most variable, with the native substrate camphor binding most tightly and camphane showing much lower affinity, as reported previously, 19,28 and the isoborneol showing intermediate affinity. For Y96CNF, the affinity for camphor is also highest, but the affinities for isoborneol and camphane are higher than those of WT.…”

Section: Resultssupporting

confidence: 80%

Conformational Heterogeneity and the Affinity of Substrate Molecular Recognition by Cytochrome P450cam

2017

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The broad and variable substrate specificity of cytochrome P450 enzymes makes them a model system for studying the determinants of protein molecular recognition. The archetypal cytochrome P450cam (P450cam) is a relatively specific P450, a feature once attributed to the high rigidity of its active site. However, increasingly studies have provided evidence of the importance of conformational changes to P450cam activity. Here we used infrared (IR) spectroscopy to investigate the molecular recognition of P450cam. Toward this goal, and to assess the influence of a hydrogen bond (H-bond) between active site residue Y96 and substrates, two variants in which Y96 is replaced by a cyanophenyl (Y96CNF) or phenyl (Y96F) group were characterized in complexes with the substrates camphor, isoborneol, and camphane. These combinations allow for a comparison of complexes in which the moieties on both the protein and substrate can serve as a H-bond donor, acceptor, or neither. The IR spectra of heme-bound CO and the site-specifically incorporated CN of Y96CNF were analyzed to characterize the number and nature of environments in each protein, both in the free and bound states. Although the IR spectra do not support the idea that protein–substrate H-bonding is central to P450cam recognition, the data altogether suggest that the differing conformational heterogeneity in the active site of the P450cam variants and changes in heterogeneity upon binding of different substrates likely contribute to their variable affinities via a conformational selection mechanism. This study further extends our understanding of the molecular recognition of archetypal P450cam and demonstrates the application of IR spectroscopy combined with selective protein modification to delineate protein–ligand interactions.

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Section: Resultssupporting

confidence: 80%

Conformational Heterogeneity and the Affinity of Substrate Molecular Recognition by Cytochrome P450cam

2017

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“…Similarly, protein dynamics are believed to be involved in the differential activity toward structurally similar substrates displayed by the relatively substrate-specific cyt P450 cam 18,35,36. In this study, the dynamics in cyt P450 cam were directly characterized using 2D IR vibrational echo spectroscopy.…”

Section: Discussionmentioning

confidence: 98%

“…To prepare the norcamphor complex, the enzyme was exchanged into buffer containing 85 mM norcamphor. All other substrates were first dissolved in ethanol to 100–700 mM before addition to the concentrated protein solution (2–3 mM) to final concentrations of 4–23 mM, such that the final protein solution contained no greater than 5% substrate-free protein35 or ethanol. The samples were clarified through 0.45 μm filters before being reduced with ten-fold molar excess sodium dithionite.…”

Section: Methodsmentioning

confidence: 99%

Protein Dynamics in Cytochrome P450 Molecular Recognition and Substrate Specificity Using 2D IR Vibrational Echo Spectroscopy

2011

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Cytochrome (cyt) P450s hydroxylate a variety of substrates that can differ widely in their chemical structure. The importance of these enzymes in drug metabolism and other biological processes has motivated the study of the factors that enable their activity on diverse classes of molecules. Protein dynamics have been implicated in cyt P450 substrate specificity. Here, 2D IR vibrational echo spectroscopy is employed to measure the dynamics of cyt P450 cam from Pseudomonas putida on fast timescales using CO bound at the active site as a vibrational probe. The substrate-free enzyme and the enzyme bound to both its natural substrate, camphor, and a series of related substrates are investigated to explicate the role of dynamics in molecular recognition in cyt P450 cam and to delineate how the motions may contribute to hydroxylation specificity. In substrate-free cyt P450 cam three conformational states are populated, and the structural fluctuations within a conformational state are relatively slow. Substrate binding selectively stabilizes one conformational state, and the dynamics become faster. Correlations in the observed dynamics with the specificity of hydroxylation of the substrates, the binding affinity, and the substrates' molecular volume suggest that motions on the hundreds of ps timescale contribute to the variation in activity of cyt P450 cam towards different substrates.

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“…Conversely, six water molecules bind when camphor is expelled from the protein (53). The spin state is an indicator of the water accessibility to the Fe III atom and of the degree of solvation of the pocket (54)(55)(56)(57)(58). Access of water molecules to the pocket is largely determined by the substrate mobility that depends on the proper hydrophobic contacts of its C-10 methyl groups with the protein residues rather than on its size and hydrogen bonding of the keto group with Tyr96 (57,58).…”

Section: Discussionmentioning

confidence: 99%

“…The spin state is an indicator of the water accessibility to the Fe III atom and of the degree of solvation of the pocket (54)(55)(56)(57)(58). Access of water molecules to the pocket is largely determined by the substrate mobility that depends on the proper hydrophobic contacts of its C-10 methyl groups with the protein residues rather than on its size and hydrogen bonding of the keto group with Tyr96 (57,58). In the crystal structures of Fe III P-450 cam (substrate) complexes with substrates that are rigidly maintained both by hydrophobic contacts and by hydrogen bonding with Tyr96 such as 1R-or 1S-camphor (49,53,59) or adamantanone (60), water molecules are absent and the iron is in the high-spin state (Figure 7c,d).…”

Section: Discussionmentioning

confidence: 99%

Conformational Relaxation in Hemoproteins: The Cytochrome P-450_c_amCase

2000

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Photodissociation of (CO)P-450(cam)(substrate) complexes was found to trigger a conformational relaxation process that interferes with ligand rebinding at temperatures as low as 140 K even though the protein conformational substates (CS(1)) remain frozen. To analyze the rebinding and relaxation kinetics, we developed a model that takes the distribution of relaxation rates explicitly into account and in which rebinding and relaxation rates are connected by a linear free energy relation. In all complexes heme relaxation occurs first and is probably faster than 100 ns even at 77 K. This is the only process found in substrate-free P-450(cam). Above 140 K and in the presence of a substrate, this initial, fast rebinding state (P) progressively relaxes to another state (P degrees ) in which rebinding is slower. The relaxation rate is independent of solvent rigidity and is governed by the protein's internal dynamics. Rebinding enthalpies in P and P degrees as well as the enthalpy shift brought about by relaxation correlate with the substrate propensity to block access to the iron site. In P degrees the barrier is higher because the substrate is closer to the heme normal and exerts more steric repulsion for CO binding. The relaxation process implies the return of substrate and heme to their ligand-free positions in which access to the heme is reduced.

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Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam

Cited by 28 publications

References 38 publications

Conformational Heterogeneity and the Affinity of Substrate Molecular Recognition by Cytochrome P450cam

Conformational Heterogeneity and the Affinity of Substrate Molecular Recognition by Cytochrome P450cam

Protein Dynamics in Cytochrome P450 Molecular Recognition and Substrate Specificity Using 2D IR Vibrational Echo Spectroscopy

Conformational Relaxation in Hemoproteins: The Cytochrome P-450_c_amCase

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Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam

Cited by 28 publications

References 38 publications

Conformational Heterogeneity and the Affinity of Substrate Molecular Recognition by Cytochrome P450cam

Conformational Heterogeneity and the Affinity of Substrate Molecular Recognition by Cytochrome P450cam

Protein Dynamics in Cytochrome P450 Molecular Recognition and Substrate Specificity Using 2D IR Vibrational Echo Spectroscopy

Conformational Relaxation in Hemoproteins: The Cytochrome P-450camCase

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Conformational Relaxation in Hemoproteins: The Cytochrome P-450_c_amCase