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The MoFe protein from Azotobacter vinelandii (AvI) and the Fe protein from Clostridium pasteurianum (CpII) form a tight complex with an association constant about 10-fold higher than that for the homologous A. vinelandii Fe protein (AvII) - AvI complex and about 100-fold higher than that for the C. pasteurianum MoFe protein (CpI) - CpII complex (1). Although the CpII-AvI nitrogenase complex is unable to reduce any substrates (2), it retains MgATP hydrolysis activity (3). Recently, it was shown that an altered A. vinelandii Fe protein (L127Δ), which appears locked in a MgATP-bound conformation, and AvI form a tight complex, and that the L127Δ Fe protein can transfer a single electron to AvI (4). In light of the fact that the L127Δ tight complex undergoes an electron transfer reaction, it seemed possible that CpII might also be able to transfer an electron to AvI. Using UV-visible absorption spectroscopy, EPR, and stopped-flow spectroscopy, it was shown that CpII is able to transfer an electron to AvI in the absence of nucleotides at a rate of 0.007 s−1. In the presence of nucleotides, the rate increased about 4-fold with MgADP and 20,000-fold with MgATP. That nucleotides increase the rate of electron transfer may indicate that conformational changes within CpII induced by the binding of MgATP or MgADP make electron transfer more facile. The EPR data also suggested that in the presence of nucleotides, the equilibrium of electron transfer within the complex is shifted toward the oxidation of the [4Fe-4S] cluster of CpII. From a Marcus analysis of the temperature dependence of the rate of electron transfer, it appeared that MgADP induced electron transfer of the true type within the CpII-AvI complex, whereas with MgATP or no nucleotides, electron transfer theory may not be applicable. |
