Evidence for a Methylammonium-Binding Site on Methylamine Dehydrogenase of Thiobacillus versutus

The nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADH_OX) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADH_OX as do the monovalent cations Cs⁺, Rb⁺, and NH₄⁺. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADH_ox by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADH_OX and methylamine a red-shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADH_OX for the substrate CH₃NH₃⁺ the substrate analogues (CH₃NH₂⁺, (CH₃)₃NH+, and (CH₃)₄N⁺, and the monovalent cations Cs⁺, Rb⁺, and NH₄⁺. Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADH_ox as a cation. The resonance Raman spectra of MADH_OX in the absence and presence of Cs⁺, NH₄⁺, and (CH₃)₃NH⁺ are very similar, except for the C=0 stretching frequencies of the o-quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 cm-1 downshifts. From these Raman results and the X-ray crystal structure, we conclude that the CH₃NH₃⁺ binding site is in close proximity to the O6 carbonyl oxygen of the TTQ.