title: Cytochrome c552 mutants: structure and dynamics at the active site probed by multidimensional NMR and vibration echo spectroscopy.
abstract: Spectrally resolved infrared stimulated vibrational echo experiments are used to measure the vibrational dephasing of a CO ligand bound to the heme cofactor in two mutated forms of the cytochrome c552 from Hydrogenobacter thermophilus. The first mutant (Ht-M61A) is characterized by a single mutation of Met61 to an Ala (Ht-M61A), while the second variant is doubly modified to have Gln64 replaced by an Asn in addition to the M61A mutation (Ht-M61A/Q64N). Multidimensional NMR experiments determined that the geometry of residue 64 in the two mutants is consistent with a non-hydrogen-bonding and hydrogen-bonding interaction with the CO ligand for Ht-M61A and Ht-M61A/Q64N, respectively. The vibrational echo experiments reveal that the shortest time scale vibrational dephasing of the CO is faster in the Ht-M61A/Q64N mutant than that in Ht-M61A. Longer time scale dynamics, measured as spectral diffusion, are unchanged by the Q64N modification. Frequency-frequency correlation functions (FFCFs) of the CO are extracted from the vibrational echo data to confirm that the dynamical difference induced by the Q64N mutation is primarily an increase in the fast (hundreds of femtoseconds) frequency fluctuations, while the slower (tens of picoseconds) dynamics are nearly unaffected. We conclude that the faster dynamics in Ht-M61A/Q64N are due to the location of Asn64, which is a hydrogen bond donor, above the heme-bound CO. A similar difference in CO ligand dynamics has been observed in the comparison of the CO derivative of myoglobin (MbCO) and its H64V variant, which is caused by the difference in axial residue interactions with the CO ligand. The results suggest a general trend for rapid ligand vibrational dynamics in the presence of a hydrogen bond donor.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Massari
authors.forename: Brian L
authors.initials: BL
authors.lastname: McClain
authors.forename: Ilya J
authors.initials: IJ
authors.lastname: Finkelstein
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Lee
authors.forename: Heather L
authors.initials: HL
authors.lastname: Reynolds
authors.forename: Kara L
authors.initials: KL
authors.lastname: Bren
authors.forename: Michael D
authors.initials: MD
authors.lastname: Fayer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D002245:Carbon Dioxide; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D020125:Mutation, Missense; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D003574:Cytochrome c Group; D008024:Ligands; D002245:Carbon Dioxide; C022302:cytochrome C-552