Control of CooA activity by the mutation at the C-terminal end of the heme-binding domain

A constitutively active mutant of a CooA, in which Met(131) was replaced by Leu, was isolated by random mutagenesis. Site-directed mutagenesis at posi tion 131 revealed that M131R-CooA was also constitutively active even in th e absence of CO and that M131P-, M131D-, and M131E-CooA were constitutively inactive regardless of the presence or absence of CO. While M131L- and M13 1E-CooA showed almost the same electronic absorption spectra as those of th e wild type in the ferric, ferrous, and GO-bound forms, M131D-CooA showed t he typical spectrum of a five-coordinate heme protein in;the ferric form. T he conformational change around the heme induced by CO binding, which trigg ers the activation of CooA, is thought to be linked to the rearrangement of the conformation around the hinge region between the heme-binding and DNA- binding domains and/or of the relative orientation of the two domains to ac tivate CooA. (C) 2000 Elsevier Science Inc. All rights reserved.