Hydrogen-deuterium exchange mass spectrometry for investigation of backbone dynamics of oxidized and reduced cytochrome P450cam.
Backbone dynamics of the camphor monoxygenase cytochrome P450(cam) (CYP101) as a function of oxidation/ligation state of the heme iron were investigated via hydrogen/deuterium exchange (H/D exchange) as monitored by mass spectrometry. Main chain amide NH hydrogens can exchange readily with solvent and the rate of this exchange depends upon, among other things, dynamic fluctuations in local structural elements. A fluxional region of the polypeptide will exchange more quickly with solvent than one that is more constrained. In most regions of the enzyme, exchange rates were similar between oxidized high-spin camphor-bound and reduced camphor- and CO-bound CYP101 (CYP-S and CYP-S-CO, respectively). However, in regions of the protein that have previously been implicated in substrate access by structural and molecular dynamics investigations, the reduced enzyme shows significantly slower exchange rates than the oxidized CYP-S. This observation corresponds to increased flexibility of the oxidized enzyme relative to the reduced form. Structural features previously found to be perturbed in CYP-S-CO upon binding of the biologically relevant effector and reductant putidaredoxin (Pdx) as determined by nuclear magnetic resonance are also more protected from exchange in the reduced state. To our knowledge, this study represents the first experimental investigation of backbone dynamics within the P450 family using this methodology.
Hamuro
Yoshitomo
Y
ExSAR Corporation, 11 Deer Park Drive, Suite 103 Monmouth Junction, NJ 08852, United States.
Molnar
Kathleen S
KS
Coales
Stephen J
SJ
OuYang
Bo
B
Simorellis
Alana K
AK
Pochapsky
Thomas C
TC
D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry*; D004926:Escherichia coli; D006859:Hydrogen / Q000737:chemistry*; D013058:Mass Spectrometry; D010957:Plasmids
D006859:Hydrogen; D003903:Deuterium; D019475:Camphor 5-Monooxygenase