Adenosyl coenzyme and pH dependence of the [4Fe-4S]2+/1+ transition in lysine 2,3-aminomutase.
5'-[N-[(3S)-3-Amino-carboxypropyl]-N-methylamino]-5(')-deoxyadenosine (azaSAM), an analog of S-adenosyl-L-methionine (SAM), was used to study the cofactor-dependent reduction of the [4Fe-4S](2+) center in lysine 2,3-aminomutase to the +1 oxidation state. azaSAM has a tertiary nitrogen in place of the sulfonium center of SAM. The analog binds to lysine 2,3-aminomutase with K(d)s of 1.4+/-0.3 microM at pH 8.0 and 2.2+/-0.6 microM at pH 6.5. Reduction of the [4Fe-4S](2+) center in the presence of this analog gives a 10K [4Fe-4S](1+) electron paramagnetic resonance (EPR) signal similar to that seen with SAM or S-adenosyl-L-homocysteine (SAH). The pH dependence of cofactor-induced reduction was examined to determine whether ionization of the tertiary nitrogen (pK(a)=7.08) might affect reduction of the [4Fe-4S](2+) center. The results show similar behavior in azaSAM and SAH, demonstrating that ionization of the aza group in azaSAM does not account for pH dependence in cofactor-dependent reduction of the [4Fe-4S](2+) center. The signal shape of the low-temperature EPR signal for the [4Fe-4S](1+) center in the SAM-induced reduction displayed a pH dependence that was not observed in the azaSAM- or SAH-induced spectra. Unique features of the signal are at a maximum at the pH activity optimum of pH 8 and are diminished as the pH is lowered or raised. These features are also absent in the spectra at all pHs examined when reduction is induced by azaSAM or SAH.
Department of Biochemistry, University of Wisconsin - Madison, 53705, USA.
Glen T
GT
Hinckley
Frank J
FJ
Ruzicka
Mark J
MJ
Thompson
G Michael
GM
Blackburn
Perry A
PA
Frey
D003067:Coenzymes / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D019751:Intramolecular Transferases / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives* / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013455:Sulfur / Q000737:chemistry
D003067:Coenzymes; D007506:Iron-Sulfur Proteins; D013455:Sulfur; D012436:S-Adenosylmethionine; D007501:Iron; D019751:Intramolecular Transferases; C021518:lysine 2,3-aminomutase