Abstract
Nitrophorins (NP) 1–7 are NO-carrying heme proteins found in the saliva of the blood-sucking insect Rhodnius prolixus. The isoform NP7 displays peculiar properties, such as an abnormally high isoelectric point, the ability to bind negatively charged membranes, and a strong pH sensitivity of NO affinity. A unique trait of NP7 is the presence of Glu in position 27, which is occupied by Val in other NPs. Glu27 appears to be important for tuning the heme properties, but its influence on the pH-dependent NO release mechanism, which is assisted by a conformational change in the AB loop, remains unexplored. Here, in order to gain insight into the functional role of Glu27, we examine the effect of Glu27 → Val and Glu27 → Gln mutations on the ligand binding kinetics using CO as a model. The results reveal that annihilation of the negative charge of Glu27 upon mutation reduces the pH sensitivity of the ligand binding rate, a process that in turn depends on the ionization of Asp32. We propose that Glu27 exerts a through-space electrostatic action on Asp32, which shifts the pKa of the latter amino acid towards more acidic values thus reducing the pH sensitivity of the transition between open and closed states.
Highlights
The heme cofactor is located inside the central cavity of the lipocalin fold, which consists of an eight-stranded antiparallel β-barrel[8,9]
The pH-dependent release mechanism is effective in NP1, NP2 and NP4, the high pH-sensitivity of NO affinity in NP7 suggests that some specific traits concur in this isoform, which is the only NP able to interact with L-α-phosphatidyl-L-serine -containing phospholipid membranes with high affinity (∼4.8 nM)[15,16]
Here we report the results of a comparative study performed on the wt NP7 and its E27Q and E27V mutants using a variety of experimental and theoretical techniques, including transient absorption (TA) spectroscopy with femtosecond to millisecond temporal coverage, X-ray crystallography and molecular dynamics (MD) simulations
Summary
The heme cofactor is located inside the central cavity of the lipocalin fold, which consists of an eight-stranded antiparallel β-barrel[8,9]. Keeping in mind the functional relevance of the conversion between closed and open states and the pH dependence of this conformational transition, the potential influence of Glu[27] in modulating the kinetics of ligand binding emerges as a challenging question for understanding the role of NP7. It is unclear whether the negative charge of Glu[27] may affect such conformational transition, and the accessibility of ligands to/from the heme pocket. Our results highlight the role of the negative charge of Glu[27] in enhancing the pH sensitivity of ligand binding
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