Assignment of the hyperfine-shifted 1H-NMR signals of the heme in the oxygen sensor FixL from Rhizobium meliloti.
BACKGROUND
[corrected] The Rhizobial oxygen sensor FixL is a hemoprotein with kinase activity. On binding of strong-field ligands, a change of the ferrous or ferric heme iron from high to low spin reversibly inactivates the kinase. This spin-state change and other information on the heme pocket have been inferred from enzymatic assays, absorption spectra and mutagenesis studies. We set out to investigate the spin-state of the FixL heme and to identify the hyperfine-shifted heme-proton signals by NMR spectroscopy.


RESULTS
Using one-dimensional NMR we directly observed the high- and low-spin nature of the met- and cyanomet-FixL heme domain, respectively. We determined the hyperfine-shifted 1H-NMR signals of the heme and the proximal histidine by one- and two-dimensional spectroscopy and note the absence of distal histidine signals.


CONCLUSIONS
These findings support the spin-state mechanism of FixL regulation. They establish that the site of heme coordination is a histidine residue and strongly suggest that a distal histidine is absent. With a majority of the heme resonances identified, one- and two-dimensional NMR techniques can be extended to provide structural and mechanistic information about the residues that line the heme pocket.
Bertolucci
C
C
Department of Chemistry and Institute for Biomolecular Science, University of South Florida, 4202 East Fowler Avenue, CHE305, Tampa, FL 33620-5250, USA. ming@chuma.cas.usf.edu
Ming
L J
LJ
Gonzalez
G
G
Gilles-Gonzalez
M A
MA
D001426:Bacterial Proteins / Q000737:chemistry*; D015374:Biosensing Techniques; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D000071677:Histidine Kinase; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010100:Oxygen / Q000032:analysis; D011522:Protons; D016962:Sinorhizobium meliloti / Q000737:chemistry
D001426:Bacterial Proteins; D006420:Hemeproteins; D008024:Ligands; D011522:Protons; D006418:Heme; D006639:Histidine; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen