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 A. Gilles-Gonzalez*
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.
 
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