NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the “belt” that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by 1H NMR spectroscopy. Wild-type NP2 has heme orientation ratio (A:B) of 1:8 at equilibrium, while wild-type NP1 has A:B ~ 1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.

The high-spin Fe(III) 1H NMR spectra of wild-type NP2 and NP1 and eighteen of their site-directed mutants, and the low-spin imidazole (ImH) and in some cases cyanide complexes of representative examples of these mutants have been investigated. It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in more detail.Figure optionsDownload as PowerPoint slideHighlights
► We study two heme proteins from the saliva of the Kissing Bug, Rhodnius prolixus:
► NP2 (heme A:B = 1:8) and NP1 (A:B = 1:1), and 19 of their site‐directed mutants.
► The mutants were chosen to find the roles of each in heme orientation and seating.
► The most important residues were found to be Phe42 and Ile 120 of NP2.