Abstract
The bacterial heme protein cytochrome ć from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 +/- 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.
Highlights
Nitric oxide (NO) acts as a second messenger in several physiological systems [1, 2], is involved in the production of several cytotoxic chemical species and nitrosative stress [3], and appears as an intermediate in the denitrification process [4]
In the present study we investigated how this unusual property of the novel proximal 5c-NO AXCP complex copy; SVD, singular value decomposition; RCCP, cytochrome cЈ from R. capsulatus; c, coordinate
The intense stretching vibration (Fe-His) at ϳ233 cmϪ1 is active in 5c-His ferrous AXCP, whereas the stretching vibration (Fe-NO) at ϳ525 cmϪ1, identified by isotopic substitution [19], is active only in the 5c-NO AXCP. Because none of these bands is present in the photoproduct spectra (Fig. 3, c–f) and there is no (Fe-NO) vibration at ϳ579 cmϪ1 ascribed to 6c-NO-His AXCP [18], we readily identify the transient species as a four-coordinate AXCP formed after NO photodissociation
Summary
Preparation of the Samples—AXCP was purified as previously described [20, 21]. The protein was concentrated to 0.47 mM in heme in MES buffer (pH 6). Time-resolved Absorption Spectroscopy—Transient spectra were recorded simultaneously to kinetics as a time-wavelength matrix data using the pump-probe laser system previously described [22] for generating the broad spectral band probe pulse. The photodissociation of NO was achieved with an excitation pulse at 564 nm whose duration was ϳ40 fs with a repetition rate of 30 Hz. The transient absorption spectrum after a variable delay between pump and probe pulses was recorded by means of a CCD detector. The same sample quartz cell (1-mm optical path length) was used for recording the equilibrium spectra and the transient absorption. The pump and probe beams were collinearly superimposed and focused on the sample by a 10-cm lens, and the optical time delay between both pulses was controlled by a motorized translation stage.
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