Abstract:
:The pH-dependent behavior of the ferric forms of two soluble truncations of Rhizobium meliloti FixL, FixL (heme and kinase domains, functional), and FixLN (heme domain) are examined by UV-visible, resonance Raman, and electron paramagnetic resonance spectroscopy. Global analysis of UV-visible data indicates that the pKa for hydroxide binding is slightly higher in FixL than in FixLN. Spectroscopic data show that high-spin and low-spin hydroxide adducts of FixLN and FixL exist in a thermal spin-state equilibrium with a significant fraction of the heme in the high spin form at room temperature. FixLN and FixL differ from myoglobin and hemoglobin in that their hemes are not fully ligated by hydroxide ion under strongly alkaline conditions. In addition to the binding of hydroxide ion, both FixLN and FixL undergo additional alkaline transitions that involve the deprotonation of tyrosine residues. FixLN contains four tyrosine residues. One has a pKa of 9.6, which is indistinguishable from that for hydroxide binding to the heme. The other three tyrosines have pKas greater than 11. At pH 11, the alkaline species react with cyanide to yield the familiar low-spin cyanide adduct. Upon reduction of the heme iron, the alkaline forms of the FixL deletion derivatives are converted to their deoxy forms. Resonance Raman spectra reveal that the Fe-His stretching vibrations of deoxyFixLN and deoxyFixL are not measurably shifted from those of their neutral counterparts. Treatment of the alkaline deoxyFixLs with O2 yields the respective oxy forms. Spectroscopic evidence indicates that the loss of activity at elevated pH cannot be attributed solely to generation of a low-spin heme hydroxide. Involvement of one or more tyrosines in signal transmission between the heme and kinase domains of FixL is proposed.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Lukat-Rodgers GS,Rexine JL,Rodgers KRdoi
10.1021/bi981439vsubject
Has Abstractpub_date
1998-09-29 00:00:00pages
13543-52issue
39eissn
0006-2960issn
1520-4995pii
bi981439vjournal_volume
37pub_type
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