Effector Roles of Putidaredoxin on Cytochrome P450cam Conformational States

Liou, Shu-Hao; Mahomed, Mavish; Lee, Young Tae; Goodin, David B.

doi:10.1021/jacs.6b04110

Cited by 36 publications

(100 citation statements)

References 72 publications

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“…28 When camphor is bound to this open form, the camphor is not well-ordered and does not bind in a productive orientation. 35 These crystal structures are consistent with our MD simulations: only when both camphor and Pdx are bound is the B′ and F/G helical regions ordered sufficiently to be observed in electron density maps.…”

Section: Discussionsupporting

confidence: 88%

Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

2017

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Previous crystal structures of cytochrome P450cam complexed with its redox partner, putidaredoxin (Pdx), shows that P450cam adopts the open conformation. It has been hypothesized that the Pdx-induced shift toward the open state frees the essential Asp251 from salt bridges with Arg186 and Lys178 so that Asp251 can participate in a proton relay network required for O2 activation. This in part explains why P450cam has such a strict requirement for Pdx. One problem with this view is that looser substrate-protein interactions in the open state may not be compatible with the observed regio- and stereoselective hydroxylation. In the present study, molecular dynamics simulations show that Pdx binding favors a conformation that stabilizes the active site and decreases camphor mobility yet retains a partially open conformation compatible with the required proton relay network. The R186A mutant which frees Asp251 in the absence of Pdx retains good enzyme activity, and the crystal structure shows that product, 5-exo-hydroxycamphor, is bound. This indicates that rupture of the Asp251-Arg186 relaxes selectivity with respect to source of electrons and enables X-ray generated reducing equivalents to support substrate hydroxylation. These combined computational and experimental results are consistent with the proposed role of Pdx in assisting the release of Asp251 from ion pairs so that it can participate in proton-coupled electron transfer.

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Section: Discussionsupporting

confidence: 88%

Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

2017

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“…58 A structure of P450cam tethered to its electron transfer partner putidaredoxin showed a similarly large change in the side chain orientation of Y96. 59 In addition, a recent structure of P450cam in the open state with camphor bound showed the Y96 side chain was disordered, 60 suggestive of high conformational heterogeneity, which agrees with the heterogeneity observed for Y96CNF in solution by IR spectroscopy.…”

Section: Discussionsupporting

confidence: 79%

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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“…Low-temperature EPR studies suggested that Pdx binding leads to a mixture of open and closed states for the oxidized cytP450cam but not for the reduced, CO-bound state (31,32). Modeling work supports a view that Pdx binding does affect the active site, enabling the catalytic reaction and perhaps leading to partial opening of the substrate access channel (33).…”

Section: Significancementioning

confidence: 82%

Identification of productive and futile encounters in an electron transfer protein complex

Andrałojć

Hiruma

Wei-min

et al. 2017

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

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Well-defined, stereospecific states in protein complexes are often in exchange with an ensemble of more dynamic orientations: the encounter states. The structure of the stereospecific complex between cytochrome P450cam and putidaredoxin was solved recently by X-ray diffraction as well as paramagnetic NMR spectroscopy. Other than the stereospecific complex, the NMR data clearly show the presence of additional states in the complex in solution. In these encounter states, populated for a small percentage of the time, putidaredoxin assumes multiple orientations and samples a large part of the surface of cytochrome P450cam. To characterize the nature of the encounter states, an extensive paramagnetic NMR dataset has been analyzed using the Maximum Occurrence of Regions methodology. The analysis reveals the location and maximal spatial extent of the additional states needed to fully explain the NMR data. Under the assumption of sparsity of the size of the conformational ensemble, several minor states can be located quite precisely. The distribution of these minor states correlates with the electrostatic potential map around cytochrome P450cam. Whereas some minor states are on isolated positively charged patches, others are connected to the stereospecific site via positively charged paths. The existence of electrostatically favorable pathways between the stereospecific interaction site and the different minor states or lack thereof suggests a means to discriminate between productive and futile encounter states.paramagnetic NMR | encounter complex | cytochrome P450cam | putidaredoxin | maximum occurrence C rystal structures suggest that proteins assume unique, stereospecific orientations within protein-protein complexes. However, a number of studies in solution have made clear that encounter states are an inherent element of protein complexes (1-8), especially in electron transfer (ET), where the interactions are often extremely fast (9). In the encounter complex, the proteins assume multiple other orientations, often in equilibrium with the major stereospecific state. In low-affinity complexes with dissociation constant (K d ) values > 10 μM, the encounter complex can represent a sizeable fraction, and in some cases, a well-defined, stereospecific complex may even be absent (10-15). The presence of encounter states may be a consequence of the chemical nature of proteins. In nonobligate stereospecific complexes, the interface represents a small fraction of the total protein surface, and therefore, it is reasonable to assume that weak interactions also occur elsewhere. In the case in which protein pairs have evolved to exhibit a high association rate by using electrostatic preorientation, electrostatic patches seem to enhance the presence of encounter states (16,17). On the one hand, the preorientation reduces the surface area that is visited by the partner, thus enhancing the number of productive encounters, but on the other hand, highly charged patches can bind the oppositely charged protein in many orientations with a...

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Effector Roles of Putidaredoxin on Cytochrome P450cam Conformational States

Cited by 36 publications

References 72 publications

Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics

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

Identification of productive and futile encounters in an electron transfer protein complex

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