Site-Directed Mutagenesis of Proline 94 to Alanine in Amicyanin Converts a True Electron Transfer Reaction into One That Is Kinetically Coupled

Publisher Website
Google Scholar
Abstract
Amicyanin is a type I copper protein that mediates electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome c-551i. Pro94 resides in the “ligand loop” of amicyanin, a sequence of amino acids that contains three of the four copper ligands. ET from the reduced O-quinol tryptophan tryptophylquinone of MADH to oxidized P94A amicyanin is a true ET reaction that exhibits values of electronic coupling (HAB) and reorganization energy (λ) that are the same as for the reaction of native amicyanin. In contrast, the parameters for the ET reaction from reduced P94A amicyanin to oxidized cytochrome c-551i have been significantly altered as a consequence of the mutation. These values of HAB and λ are 8.3 cm-1 and 2.3 eV, respectively, compared to values of 0.3 cm-1 and 1.2 eV for the reaction of native reduced amicyanin. The crystal structure of reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 Å apart [Carrell, C. J., Sun, D., Jiang, S., Davidson, V. L., and Mathews, F. S. (2004) Biochemistry43, 9372−9380]. In one of these, conformation B, a water molecule has replaced Met98 as a copper ligand, and the ET distance to the heme of the cytochrome is increased by 1.4 Å. Analysis of these structures suggests that the true kET for ET from the copper in conformation B to heme would be much less than for ET from conformation A. A novel kinetic mechanism is proposed to explain these data in which the reduction of Cu2+ by methylamine dehydrogenase is a true ET reaction while the oxidation of Cu1+ by cytochrome c-551i is kinetically coupled ET. By comparison of the temperature dependence of the observed rate of the coupled ET reaction from reduced P94A amicyanin to cytochrome c-551i with the predicted rates and temperature dependence for the true ET reaction from conformation A, it was possible to determine the Keq and values of ΔH° and ΔS° that are associated with the non-ET reaction that modulates the observed ET rate.