Nitrophorion 2 (NP2) is one of four heme proteins found in the salivary glands of the blood-sucking insect Rhodnius prolixus (also called �the kissing bug�), from the Amazon river basin. These four heme proteins bind nitric oxide (NO) in the salivary glands at low pH (5.0-6.0), thus keeping it stable for periods of up to a month by binding it to Fe(III) heme proteins. When the insect spits its saliva into the tissues of a victim during a bite, the pH rise and ~100-fold dilution cause NO to be released from the proteins to cause dilation of the capillaries in the vicinity of the bite. We are investigating the dynamics of these proteins by NMR spectroscopy, first by investigating NP2-NO as a function of pH. We find that at pH 5.0 and 6.5 the NP2 protein with NO bound is extremely rigid. NO is locked inside, and cannot escape. By pH 7.3 (the approximate pH of the victim's tissues and blood), the protein shows significant dynamics, both on the nano- to picosecond timescales, and also on the micro- to millisecond timescales, which are more appropriate to the rate constants for NO association and dissociation (kon = 3.7 � 0.5 �M-1s-1, koff = 0.093 � 0.002 s-1, Kd = 25 � 3 nM at pH 7.5). We hope, by the time of this meeting, to be able to compare the dynamics of the NO complex to those of the NO-off, high-spin, Fe(III) form of the protein over the same pH range.

Dynamics of native N-terminus NP2 bound to nitric oxide, as studied by NMR spectroscopy

244th National Meeting (2012)

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