Assignment of Hyperfine-Shifted 1H NMR Signals of the Heme Pocket in the Oxygen Sensor FixL Kinase from Rhizobium meliloti
Craig Bertolucci and Li-June Ming*
Department of Chemistry and Institute for Biomolecular Science
University of South Florida
Tampa, Florida 33620-5250
Gonzalo Gonzalez and Marie
Department of Microbiology and Plant Biotechnology Center
The Ohio State University
Columbus, Ohio 43210-1002
Received May 21, 1996; Accepted July 2, 1996
Background: 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
reversible inactivates the kinase. The 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 indentify 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.
Reprint in PDF
(Back to Current Publications.)