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Chem Biol. 1996 Jul;3(7):561-6.

Assignment of the hyperfine-shifted 1H-NMR signals of the heme in the oxygen sensor FixL from Rhizobium meliloti.

Author information

1
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

Abstract

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.

PMID:
8807888
[Indexed for MEDLINE]
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