Study of ion translocation by respiratory complex I. A new insight using Na-23 NMR spectroscopy

The research on complex I has gained recently a new enthusiasm, especially after the resolution of the crystallographic structures of bacterial and mitochondrial complexes. Most attention is now dedicated to the investigation of the energy coupling mechanism(s). The proton has been identified as the coupling ion, although in the case of some bacterial complexes I Na+ has been proposed to have that role. We have addressed the relation of some complexes I with Na+ and developed an innovative methodology using Na-23 NMR spectroscopy. This allowed the investigation of Na+ transport taking the advantage of directly monitoring changes in Na+ concentration. Methodological aspects concerning the use of Na-23 NMR spectroscopy to measure accurately sodium transport in bacterial membrane vesicles are discussed here. Externalvesicle Na+ concentrations were determined by two different methods: 1) by integration of the resonance frequency peak and 2) using calibration curves of resonance frequency shift dependence on Na+ concentration. Although the calibration curves are a suitable way to determine Na+ concentration changes under conditions of fast exchange, it was shown not to be applicable to the bacterial membrane vesicle systems. In this case, the integration of the resonance frequency peak is the most appropriate analysis for the quantification of external-vesicle Na+ concentration.