Conformational role of the divalent metal in bovine heart mitochondrial F1-ATPase: an electron spin echo envelope modulation study.
The catalytic sites of beef heart mitochondrial F1-ATPase were studied by electron spin echo envelope modulation (ESEEM) spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II), maintaining the enzyme catalytic activity. F1-ATPase was purified from beef heart mitochondria. A protein still containing three endogenous nucleotides, named MF1(1,2), is obtained under milder conditions, whereas a harsher treatment gives a fully depleted F1, named MF1(0,0). Several samples were prepared, loading MF1(0,0) or MF1(1,2) with Mn(II) or MnIIADP in both substoichiometric and excess amounts. When MF1(1,2) is loaded with Mn(II) in a 1:0.8 ratio, the FT-ESEEM spectrum shows evidence of a nitrogen interacting with the metal, while this interaction is not present in MF1(0,0) + Mn(II) in a 1:0.8 ratio. However, when MF1(0,0) is loaded with 2.4 Mn(II), the FT-ESEEM spectrum shows a metal-nitrogen interaction resembling that present in MF1(1,2) + Mn(II) in a 1:0.8 ratio. These results strongly support the role of the metal alone in shaping and structuring the catalytic sites of the enzyme. When substoichiometric ADP is added to MF1(1,2) preloaded with 0.8 equiv of Mn(II), the ESEEM spectra show evidence of a phosphorus nucleus coupled to the metal, indicating that the nucleotide phosphate binding to Mn(II) occurs in a catalytic site. Generally, 14N coordination to the metal is clearly identified in the ESEEM spectra of all the samples containing more than one metal equivalent. One point of note is that the relevant nitrogen-containing ligand(s), responsible for the signals in the ESEEM spectra, has not yet been identified in the available X-ray structures.
Zoleo
Alfonso
A
Department of Chemistry, University of Padova, via Marzolo 1, Italy.
Lippe
Giovanna
G
Contessi
Stefania
S
Brustolon
Marina
M
Dabbeni-Sala
Federica
F
Maniero
Anna Lisa
AL
D000818:Animals; D002413:Cations, Divalent / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation
D002413:Cations, Divalent; D008345:Manganese; D025261:Mitochondrial Proton-Translocating ATPases