Redox-Dependent Dynamics in Cytochrome P450cam

• Published in: Biochemistry (Easton) 2009-05, Vol.48 (20), p.4254-4261
• Main Authors: Pochapsky, Susan Sondej, Dang, Marina, OuYang, Bo, Simorellis, Alana K, Pochapsky, Thomas C
• Format: Article
• Language: English
• Subjects: Camphor 5-Monooxygenase - chemistry; Catalytic Domain; Cytochromes - chemistry; Enzymes - chemistry; Escherichia coli - metabolism; Ferredoxins - chemistry; Iron-Sulfur Proteins - chemistry; Magnetic Resonance Spectroscopy - methods; Mass Spectrometry - methods; Models, Molecular; Molecular Conformation; Oxidation-Reduction; Oxygen - chemistry; Pseudomonas - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi900002k

Local protein backbone dynamics of the camphor hydroxylase cytochrome P450cam (CYP101) depend upon the oxidation and ligation state of the heme iron. 1H−15N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen−deuterium exchange kinetics (H−D exchange) and 15N relaxation measurements, and these results are compared with previously published results obtained by H−D exchange mass spectrometry. In general, the reduced enzyme exhibits lower-amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B′ helix and β3 and β5 sheets) and binding of putidaredoxin (C and L helices), the iron−sulfur protein that acts as the effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and the relevance of the observed effects to the enzyme mechanism is discussed.