Re-engineering monovalent cation binding sites of methylamine dehydrogenase: Effects on spectral properties and gated electron transfer

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-de pendent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from sub strate-reduced MADH to amicyanin. Two putative monovalent cation binding si tes in MADH have been identified by X-ray crystallography [Labesse, G., Fer rari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathew s, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi in teractions involving residue alpha Phe55. An alpha F55A mutation differenti ally affects these two monovalent cation-dependent phenomena. The apparent K-d associated with spectral perturbations increases 10-fold. The apparent K-d associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the require ment for the cation for the gated ET. These results show that of the two bi nding sites revealed in the structure, cation binding to the distal site, w hich is stabilized by the cation-pi interactions, is responsible for the sp ectral perturbations. Cation binding to the proximal site, which is stabili zed by several oxygen ligands, is responsible for the enhancement of the ra te of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited ca tion binding properties that were the same as those of the native enzyme, i ndicating that interactions with the carboxylate of Glu can effectively rep lace the cation-pi interactions with Phe in stabilizing monovalent cation b inding to the distal site.