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The molecular basis of formation of a complex of methylamine dehydrogenase (MADH) and amicyanin has been studied using inverse mutagenesis of an interprotein salt bridge. This is the first site-directed mutagenesis of MADH and the first construction, heterologous expression and characterization of a six-His-tagged MADH. The complex is necessary for interprotein electron transfer. The salt bridge is between Arg99 of amicyanin and Asp180 of the alpha subunit of MADH. A model that may also be usable for looking at the sequence of events leading to stable MADH-amicyanin complex formation has been suggested.

The two-electron oxidation of tryptophan tryptophylquinone (TTQ) in substrate-reduced methylamine dehydrogenase (MADH) by amicyanin occurs through an N-semiquinone intermediate in which the substrate-derived amino group remains covalently attached to TTQ. A new technique for the stoichiometric formation of the N-semiquinone in vitro was used to investigate the oxidation of the N-semiquinone by amicyanin in much more detail than previously possible.

The molecular aspects of interprotein complex-dependent change in the redox potential of amicyanin were investigated. It was found that this effect can be largely attributed to the significant pH reduction of the pK(subscript a) value of lower amounts of amicyanin as a result of complex formation with the quinoprotein methylamine dehydrogenase. Thus, it can be concluded that protein-protein interactions substantially affect the redox characteristics of type I copper protein amicyanin by restricting a pH-dependent, redox-based conformational change of one of the copper ligands.

A study was conducted to test whether the Phe 97 of amicyanin to Glu mutation affects the rate constant for the electron transfer reaction from quinol methylamine dehydrogenase (MADH) to amicyanin. Quinol MADH was combined with various concentrations of F97E mutant amicyanins that was represented as a function of temperature dependence of the electron transfer rate constant. Results showed that Phe 97 of amicyanin to Glu influences complex formation with MADH.

Research reveals that mutation in alpha-phenylalanine55 of the methylamine dehydrogenase alters its substrate preference to primary amine with at least seven carbons. Data also show that the enzyme reaction rate is substantially reduced compared to the wild-type.

Proteins bind redox cofactors, modifying their electrochemistry and affinity by specific interactions of the binding site with each cofactor redox state. Photosynthetic reaction centers from Rhodobacter sphaeroides have three ubiquinone-binding sites, Q(A), and proximal and distal Q(B) sites. Ubiquinones, which can be doubly reduced and bind 2 protons, have 9 redox states. However, only Q and Q(-) are seen in the Q(A) site and Q, Q(-), and QH(2) in the proximal Q(B) site. The distal Q(B) function is uncertain. Multiple conformation continuum electrostatics (MCCE) was used to compare the ubiquinone electrochemical midpoints (E(m)) and pK(a) values at these three sites. At pH 7, the Q(A)/Q(A)(-) E(m) is -40 mV and proximal Q(B)/Q(B)(-) -10 mV in agreement with the experimental values (assuming a solution ubiquinone E(m) of -145 mV). Q(B) reduction requires changes in nearby residue protonation and SerL223 reorientation. The distal Q(B)/Q(B)(-) E(m) is a much more unfavorable -260 mV. Q(A) and proximal Q(B) sites generally stabilize species with a -1 charge, while the distal Q(B) site prefers binding neutral species. In each site, the dianion is destabilized because favorable interactions with the residues and backbone increase with charge (q), while the unfavorable loss of solvation energy increases with q(2). Therefore, proton binding before a second reduction, forming QH and then QH(-), is always preferred to forming the dianion (Q(-)(2)). The final product QH(2) is higher in energy at the proximal Q(B) site than in solution; therefore, it binds poorly, favoring release. In contrast, QH(2) binds more tightly than Q at the distal Q(B) site.