Characterization of two Cu-containing protein fragments obtained by limited proteolysis of Hyphomicrobiumdenitrificans A3151 nitrite reductase

The unusual Hyphomicrobium denitrificans nitrite reductase containing two type 1 Cu sites and one type 2 Cu site (MW, 50 kDa) has been proteolyzed to two protein fragments (14 and 35 kDa) with subtilisin. The visible absorption, CD, and EPR spectra of these proteins imply that the blue 14-kDa protein fragment has one type 1 Cu site, which is axially elongated trigonal bipyramidal, and the green 35-kDa protein fragment has one type 1 Cu site having a flattened tetrahedral geometry with one type 2 Cu site. The 35-kDa fragment shows the nitrite reduction activity a little higher than to that of native HdNIR. The redox potentials of the 14- and 35-kDa fragments are +345 and +353mV vs. NHE at pH 7.0, respectively. Moreover, the intermolecular electron transfer rate constant of the 35-kDa fragment from an electron donor, cognate cytochrome c(550), is nearly the same as that of the native enzyme.