Binding sites for Mg(II) in H(+)-ATPase from Bacillus PS3 and in the alpha 3 beta 3 gamma subcomplex studied by one-dimensional ESEEM and two-dimensional HYSCORE spectroscopy of oxovanadium(IV) complexes: a possible role for beta-His-324.
The binding sites for Mg2+ in wild type F1 ATPase (TF1) and in the alpha 3 beta 3 gamma subcomplex from the thermophilic bacterium Bacillus PS3 have been studied by EPR and by ESEEM and HYSCORE spectroscopy of complexes with the oxovanadium cation VO2+. Complexes of metal-depleted TF1 and substoichiometric amounts of VO2+ display low-temperature EPR signals with spectral parameters g parallel = 1.947 and g perpendicular = 1.980, and hyperfine couplings with 51V, A parallel = 169 x 10(-4) cm-1 and A perpendicular = 61 x 10(-4) cm-1, that are indicative of a binding site for VO2+ with nitrogen ligands from the protein. This binding site is probably identical with the metal binding site with strong affinity M1 that has been characterized using Mn2+ in a previous study [Buy, C., Girault, G., & Zimmermann, J. L. (1996) Biochemistry 35, 9880-9891]. The three-pulse ESEEM spectrum of the VO2+ complex with TF1 shows a frequency pattern with spectral properties that are evidence for two nitrogen ligands to the VO2+ with hyperfine couplings A1 = 4.75 MHz and A2 = 6.5 MHz and nuclear quadrupole parameters e2Qq1 = 2.8-3.2 MHz and e2Qq2 = 2.0-2.3 MHz. The ligands are identified as a lysine terminal amine and a histidine imidazole, which are proposed as Lys-164 and His-324 from a beta subunit. The HYSCORE data obtained for the VO.TF1 complex show correlations within each pair of the ESEEM nu dq peaks from the 14N nuclei, confirming the interpretation of the one-dimensional spectra. Evidence for the formation of a ternary complex by addition of VO2+ and ATP to metal-depleted TF1 is shown in the EPR and ESEEM spectra and in the contour plots of the HYSCORE data. Two pairs of correlation patterns are resolved in addition to the peaks from the two 14N ligands, which are interpreted as hyperfine couplings with 31P beta and 31P gamma of the ATP that binds the VO2+ cation. The assignment of the two hyperfine couplings to the specific phosphates, A(31P beta) = 15.5 MHz and A(31P gamma) = 8.7 MHz, in the VO.TF1.ATP complex is proposed by comparison with those measured for VO2+ in solution with ATP at pH 6.3 and 2.3. These results are discussed in light of the previous data with the analogous Mn.TF1 complex, and a model is proposed in which the native Mg2+ in the M1 site is coordinated by the side chain of beta-Lys-164 and is in close proximity to a histidine residue (probably beta-His-324) that may have a critical role. Additional coordination by two phosphates from ATP (probably the beta- and gamma-phosphates) is observed in the ternary complex VO.TF1.ATP. ESEEM and HYSCORE data are also obtained for the analogous complexes VO. alpha 3 beta 3 gamma and VO. alpha 3 beta 3 gamma .ATP that show very similar properties in terms of coordination of the divalent metal cation, except for the lysine ligand that is found to be lost in the ternary complex with ATP. It is suggested that this observation may reflect changes in the metal and nucleotide active sites that are associated with the absence of the delta and epsilon subunits in the subcomplex.
Buy
C
C
Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, Gif-sur-Yvette, France.
Matsui
T
T
Andrianambinintsoa
S
S
Sigalat
C
C
Girault
G
G
Zimmermann
J L
JL
D000255:Adenosine Triphosphate / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005998:Glycine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D014638:Vanadates / Q000737:chemistry
D011994:Recombinant Proteins; C075743:oxovanadium IV; D014638:Vanadates; D008345:Manganese; D006639:Histidine; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium; D005998:Glycine