Amicyanin Transfers Electrons from Methylamine Dehydrogenase to Cytochrome c-551i via a Ping-Pong Mechanism, not a Ternary Complex

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Abstract

The first crystal structure of a ternary redox protein complex was comprised of the enzyme methylamine dehydrogenase (MADH) and two electron transfer proteins, amicyanin and cytochrome c-551i from Paracoccus denitrificans [Chen et al. Science 1994, 264, 86-90]. The arrangement of the proteins suggested possible electron transfer from the active site of MADH via the amicyanin copper ion to the cytochrome heme iron, although the distance between the metals is large. We studied the interactions between these proteins in solution. A titration followed by NMR spectroscopy shows that amicyanin binds cytochrome c-551i. The interface comprises the hydrophobic and positive patches of amicyanin, not the binding site observed in the ternary complex. NMR experiments further show that amicyanin binds tightly to MADH with an interface that matches the one observed in the crystal structure and that mostly overlaps with the binding site for cytochrome c-551i. Upon addition of cytochrome c-551i, no changes in the NMR spectrum of MADH-bound amicyanin are observed, suggesting that a possible interaction of the cytochrome with the binary complex must be very weak, with a dissociation constant higher than 2 mM. Reconstitution of the entire redox chain in vitro demonstrates that amicyanin can react rapidly with cytochrome c-551i, but that association of amicyanin with MADH inhibits this reaction. It is concluded that electron transfer from MADH to cytochrome c-551i does not involve a ternary complex but occurs via a ping-pong mechanism in which amicyanin uses the same interface for the reactions with MADH and cytochrome c-551i.

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