Abstract
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 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.
Original language | English (US) |
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Pages (from-to) | 561-566 |
Number of pages | 6 |
Journal | Chemistry and Biology |
Volume | 3 |
Issue number | 7 |
DOIs | |
State | Published - Jul 1996 |
Keywords
- FixL
- NMR
- myoglobin
- nitrogen fixation
- sensor kinase
ASJC Scopus subject areas
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Pharmacology
- Drug Discovery
- Clinical Biochemistry