Ultrafast dynamics of diatomic ligand binding to nitrophorin 4

Abstract

Nitrophorin 4 (NP4) is a heme protein that stores and delivers nitric oxide (NO) through pH-sensitive conformational change. This protein uses the ferric state of a highly ruffled heme to bind NO tightly at low pH and release it at high pH. In this work, the rebinding kinetics of NO and CO to NP4 are investigated as a function of iron oxidation state and the acidity of the environment. The geminate recombination process of NO to ferrous NP4 at both pH 5 and pH 7 is dominated by a single approximately 7 ps kinetic phase that we attribute to the rebinding of NO directly from the distal pocket. The lack of pH dependence explains in part why NP4 cannot use the ferrous state to fulfill its function. The kinetic response of ferric NP4NO shows two distinct phases. The relative geminate amplitude of the slower phase increases dramatically as the pH is raised from 5 to 8. We assign the fast phase of NO rebinding to a conformation of the ferric protein with a closed hydrophobic pocket. The slow phase is assigned to the protein in an open conformation with a more hydrophilic heme pocket environment. Analysis of the ultrafast kinetics finds the equilibrium off-rate of NO to be proportional to the open state population as well as the pH-dependent amplitude of escape from the open pocket. When both factors are considered, the off-rate increases by more than an order of magnitude as the pH changes from 5 to 8. The recombination of CO to ferrous NP4 is observed to have a large nonexponential geminate amplitude with rebinding time scales of approximately 10(-11)-10(-9) s at pH 5 and approximately 10(-10)-10(-8) s at pH 7. The nonexponential CO rebinding kinetics at both pH 5 and pH 7 are accounted for using a simple model that has proven effective for understanding CO binding in a variety of other heme systems (Ye, X.; et al. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 14682).

Figure 1

Rebinding kinetics of NO to ferrous NP4 (pumped at 403 nm and probed at 438 nm) at pH 5.0 and pH7.0. The solid lines represent the fits of the data using a two exponential function. The kinetic traces are normalized to unity at time zero.

Figure 2

Transient absorption spectra of the photolyzed ferric NP4NO at pH 5.6, for different time delays. The pump excitation wavelength was 580 nm.

Figure 3

Rebinding kinetics of NO to ferric NP4 (pumped at 403 nm and probed at 420 nm) at pH 5.0 and pH8.0. The solid lines represent the fits to the data using the kinetic model developed in the text. The kinetic traces are normalized to unity at time zero.

Figure 4

Spectra used for calculation of photolysis quantum yield of ferric NP4NO. (A) Spectra of ferric NP4NO at pH 5.6, (B) Spectra of MbCO. In both figures (A) and (B) the red, black and blue lines represent, respectively, the transient absorption spectrum at t = 20 ps, the scaled equilibrium spectrum of the reactant, and the calculated spectrum of the 5-coordinate photoproduct at t = 20 ps.

Figure 5

Kinetic traces of CO rebinding to NP4 at pH 7.0 and pH 5.0. (pumped at 403 nm and probed at 438 nm). The solid lines are fits using the heme doming distribution model: The red line represents a fit function that assumes a separate distribution for each of the open and closed states. The green line represents a fit function with only a single “average” distribution. The data are normalized to unity at time zero. The inset represents the rebinding kinetics of CO to NP4 at pH 7.0 over a wider time window. The slower bimolecular phase has a ~25% amplitude.

Figure 6

Transient absorption spectrum of ferric NP4NO at t =20 ps (red), the scaled equilibrium spectrum of NP4NO (black), the calculated spectrum of the 5-coordinate photoproduct (blue) and the scaled equilibrium spectrum of 6-coordinate NP4-H2O (magenta).

Scheme 1