| Abstract
| - The catalytic sites of beef heart mitochondrial F1-ATPase were studied by electron spin echoenvelope modulation (ESEEM) spectroscopy, using Mn(II) as a paramagnetic probe, which replaces thenaturally occurring Mg(II), maintaining the enzyme catalytic activity. F1-ATPase was purified from beefheart mitochondria. A protein still containing three endogenous nucleotides, named MF1(1,2), is obtainedunder milder conditions, whereas a harsher treatment gives a fully depleted F1, named MF1(0,0). Severalsamples were prepared, loading MF1(0,0) or MF1(1,2) with Mn(II) or MnIIADP in both substoichiometricand excess amounts. When MF1(1,2) is loaded with Mn(II) in a 1:0.8 ratio, the FT-ESEEM spectrumshows 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 spectrumshows a metal−nitrogen interaction resembling that present in MF1(1,2) + Mn(II) in a 1:0.8 ratio. Theseresults strongly support the role of the metal alone in shaping and structuring the catalytic sites of theenzyme. When substoichiometric ADP is added to MF1(1,2) preloaded with 0.8 equiv of Mn(II), theESEEM spectra show evidence of a phosphorus nucleus coupled to the metal, indicating that the nucleotidephosphate binding to Mn(II) occurs in a catalytic site. Generally, 14N coordination to the metal is clearlyidentified in the ESEEM spectra of all the samples containing more than one metal equivalent. One pointof 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.
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