Dimerization of nitrophorin 4 at low pH and comparison to the K1A mutant of nitrophorin 1.

Nitrophorin 4, one of the four NO-carrying heme proteins from the salivary glands of Rhodnius prolixus, forms a homodimer at pH 5.0 with a Kd of ∼8 µM. This dimer begins to dissociate at pH 5.5 and is completely dissociated to monomer at pH 7.3, even at 3.7 mM. The dimer is significantly stabilized by binding NO to the heme and at pH 7.3 would require dilution to well below 0.2 mM to completely dissociate the NP4-NO homodimer. The primary techniques used for investigating the homodimer and the monomer-dimer equilibrium were size-exclusion fast protein liquid chromatography at pH 5.0 and (1)H{(15)N} heteronuclear single-quantum coherence spectroscopy as a function of pH and concentration. Preparation of site-directed mutants of NP4 (A1K, D30A, D30N, V36A/D129A/L130A, K38A, R39A, K125A, K125E, D132A, L133V, and K38Q/R39Q/K125Q) showed that the N-terminus, D30, D129, D132, at least one heme propionate, and, by association, likely also E32 and D35 are involved in the dimerization. The "closed loop" form of the A-B and G-H flexible loops of monomeric NP4, which predominates in crystal structures of the monomeric protein reported at pH 5.6 but not at pH 7.5 and which involves all of the residues listed above except D132, is required for dimer formation. Wild-type NP1 does not form a homodimer, but NP1(K1A) and native N-terminal NP1 form dimers in the presence of NO. The homodimer of NP1, however, is considerably less stable than that of NP4 in the absence of NO. This suggests that additional aspartate or glutamate residues present in the C-terminal region of NP4, but not NP1, are also involved in stabilizing the dimer.

NMR investigations of nitrophorin 2 belt side chain effects on heme orientation and seating of native N-terminus NP2 and NP2(D1A).

Nitrophorin 2 (NP2), one of the four NO-storing and NO-releasing proteins found in the saliva of the blood-sucking bug Rhodnius prolixus, has a more ruffled heme and a high preference for a particular heme orientation (B) compared with nitrophorin 1 and nitrophorin 4, which show not a preference (A to B ratio of approximately 1:1), suggesting that it fits more tightly in the ß-barrel protein. In this work we have prepared a series of "belt" mutants of NP2(D1A) and (ΔM0)NP2 aimed at reducing the size of aromatic or other residues that surround the heme, and investigated them as the high-spin aqua and low-spin N-methylimidazole complexes. The belt mutants included Y38A, Y38F, F42A, F66A, Y85A, Y85F, Y104A, I120T, and a triple mutant of NP2(D1A), the F42L, L106F, I120T mutant. Although I120 has been mainly considered to be a distal pocket residue, CδH3 of I120 lies directly above the heme 3-methyl, at 2.67 Å, of heme orientation B, or the 2-vinyl of A, and it thus plays a role as a belt mutant, a role that turns out to be extremely important in creating the strong favoring of the B heme orientation [A to B ratio of 1:14 for NP2(D1A) or 1:12 for (ΔM0)NP2]. The results show that the 1D (1)H NMR spectra of the high-spin forms are quite sensitive to changes in the shape of the heme binding cavity. The single mutation I120T eliminates the favorability of the B heme orientation by producing a heme A to B orientation ratio of 1:1, whereas the single mutation F42A reverses the heme orientation from an A to B ratio of 1:14 seen for NP2(D1A) to 10:1 for NP2(D1A,F42A). The most extreme ratio was found for the triple mutant of NP2(D1A), NP2(D1A,F42L,L105F,I120T), in which the A to B ratio is approximately 25:1, a ΔG change of about -3.5 kcal/mol or -14.1 kJ/mol with respect to NP2(D1A). The seating of the heme is modified as well in that mutant and in several others, by rotations of the heme by up to 4° from the seating observed in NP2(D1A), in order to relieve steric interactions between a vinyl ß-carbon and a protein side chain, or to fill a cavity created by replacing a large protein side chain by a much smaller one; the latter was observed for all tyrosine to alanine mutants. These relatively small changes in seating have a measurable effect on the NMR spectra of the mutants, but are indeed minor in terms of overall seating and reactivity of the NP2(D1A) protein. The (1)H NMR resonances of the hemin substituents of the low-spin N-methylimidazole complexes of NP2(D1A,F42L,L105F,I120T) as well as NP2(D1A,I120T), NP2(D1A,Y104A), and NP2(D1A,F42A) have been assigned using natural abundance (1)H{(13)C} heteronuclear multiple quantum correlation and (1)H-(1)H nuclear Overhauser effect spectroscopy spectra.