Resonance Raman spectra of an O2-binding H-NOX domain reveal heme relaxation upon mutation.
Resonance Raman spectra were measured for the wild type Heme-Nitric oxide/OXygen binding domain from Thermoanaerobacter tengcongensis (Tt H-NOX WT) and three other Tt H-NOX proteins containing mutations at key conserved residues to determine the heme conformation in solution. The most dramatic changes in heme conformation occurred in the O2-bound forms, and the single Tt H-NOX P115A mutation was sufficient to generate a significant relaxation of the chromophore. Clear evidence of heme relaxation in the Tt H-NOX I5L, P115A, and I5L/P115A mutants in solution is demonstrated by the observation of reduced resonance Raman intensities for several out-of-plane low frequency modes (e.g., gamma11, gamma12, gamma13, and gamma15) in the 400-750 cm(-1) region known to be sensitive to ruffling and saddling deformations, as well as increased vibrational frequencies for the core heme skeletal stretching modes, nu3, nu2, and nu10. In addition, all three mutants exhibited some degree of heme conformational heterogeneity based on several broad skeletal markers (e.g., nu10) in the high frequency region. These results are comparable to those observed by Olea et al. for Tt H-NOX P115A in crystal form, where four different heme structures were determined from a single unit cell. On the basis of the resonance Raman spectra, it is clear that the actual heme conformation for Tt H-NOX P115A in solution is considerably more relaxed than that of the WT protein, with increased flexibility within the protein pocket, allowing for rapid sampling of alternate conformations.
Tran
Rosalie
R
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Boon
Elizabeth M
EM
Marletta
Michael A
MA
Mathies
Richard A
RA
D019943:Amino Acid Substitution / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000235:genetics; D008024:Ligands; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011392:Proline / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics; D013059:Spectrum Analysis, Raman; D046968:Thermoanaerobacter
D001426:Bacterial Proteins; D006420:Hemeproteins; D008024:Ligands; D007532:Isoleucine; D009569:Nitric Oxide; D011392:Proline; D010100:Oxygen