Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy

Krugilik et al. 10.1073/pnas.0700445104.

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SI Figure 6
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SI Figure 6

Fig. 6. Transient absorption kinetics at 421 nm of FixLH-O2 (red) and MbO2 (black).





SI Text

To investigate whether rebinding of O2 to FixLH may occur on the timescale <0.5 ps that is not accessible by transient Raman spectroscopy, transient absorption experiments were performed using the setup described in ref. 1, with a 30-fs compressed pump pulse centered at 560 nm. The chirp in the white-light continuum probe pulse was optimally compensated for at 421 nm, near the maximum of the FixLH-O2 absorption, to minimize the cross phase modulation artifact around t = 0 to <10% of the signal at this wavelength. SI Fig. 6 shows the kinetics upon excitation of FixLH-O2 and MbO2 at this wavelength. Clearly, the pump-induced bleaching rapidly decays initially. For both proteins, the initial decay was fitted with a time constant of 80 fs. The extent of this decay is significantly higher in FixLH-O2.

After the initial decay, the remaining signal on the >0.5 ps timescale is due in majority to hot 6-c heme for FixLH-O2 with a minor contribution (~5%, see main text) of 5-coordinate heme, that is not detectable in our TR3 experiments. For MbO2 the remaining signal contains a higher contribution of 5-c heme (~30%; ref. 2). Thus, the higher yield of the 80-fs phase in FixLH-O2 is correlated with a lower yield of dissociated 5-c heme after this phase; and therefore may be associated with recombination of heme and O2 following initial high-yield (possibly close to 100% for both systems) dissociation. This recombination is faster than the timescale in which doming is thought to occur (~200 fs; see main text). Therefore, in this view, recombination occurs with a 5-c planar (nondomed) heme. The electronic spectrum of such a state is unknown; therefore a more quantitative and full spectral analysis will be the subject of a future study. Nevertheless experiments performed with optimal chirp compensation at other wavelengths indicate that the initial spectra of FixLH-O2 and MbO2 are similar. In previous femtosecond experiments (2, 3) on MbO2 and HbO2 the 80-fs phase was not identified as a separate decay phase, presumably due to the lower temporal resolution and possibly contributions of the cross phase modulation artifact.

1. Jasaitis, A., Hola, K., Bouzhir-Sima, L., Lambry, J.-C., Balland, V., Vos, M. H., & Liebl, U. (2006) Biochemistry 45, 6018-6026.

2. Ye, X., Demidov, A., & Champion, P. M. (2002) J. Am. Chem. Soc. 124, 5914-5924.

3. Petrich, J. W., Poyart, C., & Martin, J.-L. (1988) Biochemistry 27, 4049-4060.

This Article

  1. Published online before print April 19, 2007, doi: 10.1073/pnas.0700445104 PNAS May 1, 2007 vol. 104 no. 18 7408-7413
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