Electron transfer in crystals of the binary and ternary complexes of methylamine dehydrogenase with amicyanin and cytochrome c551i as detected by EPR spectroscopy.

EPR studies of the methylamine dehydrogenase (MADH)-amicyanin and MADH-amicyanin-cytochrome c551i crystalline complexes have been performed on randomly oriented microcrystals before and after exposure to the substrate, methylamine, as a function of pH. The results show that EPR signals from the redox centers present in the various proteins can be observed simultaneously. These results complement and extend earlier studies of the complexes under similar conditions that utilized single-crystal polarized absorption microspectrophotometry. The binary complex shows a blue copper axial signal, characteristic of oxidized amicyanin. After reaction of substrate with the MADH coenzyme tryptophan tryptophylquinone (TTQ), the binary complex exhibits an equilibrium mixture of oxidized copper/reduced TTQ and reduced copper/TTQ. radical, whose ratio is dependent on the pH. In the oxidized ternary complex, the same copper axial signal is observed superimposed on the low-spin ferric heme features characteristic of oxidized cytochrome c551i. After addition of substrate to the ternary complex, a decrease of the copper signal is observed, concomitant with the appearance of the radical signal derived from the semiquinone form of TTQ. The equilibrium distribution of electrons between TTQ and copper as a function of pH is similar to that observed for the binary complex. This result was essential to establish that the copper center retains its function within the crystalline ternary complex. At high pH, with time the low-spin heme EPR features disappear and the spectrum indicates that full reduction of the complex by substrate has occurred.