Ultrafast dynamics of ligand-heme interaction in eNOS oxygenase domain
Chih-Chang Hung1*, Atsushi Yabushita1, Takayoshi Kobayashi1,2,3,4, Pei-Feng Cheng6, Keng S. Liang5,6
1Department of Electrophysics, National Chiao Tung University, Hsinchu, Taiwan
2Department of Applied Physics and Chemistry and Institute for Laser Science, The University of Electrocommunications, Tokyo, Japan
3CREST, JST, Saitama, Japan
4Institute of Laser Engineering, Osaka University, Osaka, Japan
5Department of Physics, National Chiao Tung University, Hsinchu, Taiwan
6Institute of Physics, Academia Sinica, Taipei, Taiwan
* presenting author:Hung ChihChang, email:hone918@gmail.com
Nitric oxide synthases (NOS) catalyze the formation of NO from oxygen and L-arginine (L-Arg). Three major isoforms, iNOS, eNOS, and nNOS, have been found in macrophages, endothelial cells and neuronal tissues, respectively. NO production by iNOS, eNOS and nNOS is important function in mammals to control plethora cellular activities. All three NOS isoforms are dimeric. Each subunit of dimer contains two domains: a reductase domain and an oxygenase domain that contains heme and tetrahydrobiopterin (H4B). The catalytic process occurs in the heme active site of the oxygenase domain. Because of the biological importance of the NOS, NOS and NOS-like proteins have been widely studied in various groups. Nevertheless, the origins of the different functionalities of these proteins are still not-fully understood even though they are conventionally studied in their stationary spectra or their quasi-stationary spectra in millisecond time-scales. Previously, the dynamics of the NOS proteins were partially studied by picosecond time-resolved absorption at a few probe wavelengths [1,2]. The information obtained there is not enough to understand the mechanism of the NOS protein dynamics.

In this work, the ultrafast dynamics of ligand-heme interaction in eNOS oxygenase domain have been studied by measuring the transient absorption change in the time region up to few ps with < 100 fs time resolution. The pump wavelength was centered at 400 nm overlapping the Soret band of heme, which is NO binding site of eNOS. The transient absorption spectra was probed at 400 nm with bandwidth of 20 nm. Thus, the time-resolved signal observed after the photo-excitation would reflect the dynamics of photo-dissociation and recombination from the heme domain of eNOS protein binding with L-Arginine or imidazole. The results of transient trace monitored at 400 nm for both samples has shown a positive absorption change of induced absorption decaying with time faster than 1 ps. It is due to the transition from the Soret band to the electronic ground state being explained by ligand rebinding to heme site of eNOS oxygenase domain. The negative components of transient absorption change decaying in range from 10 ps to 100ps of eNOS and eNOS with imidazole reflects the transition from Soret band to Q band in heme of eNOS oxygenase domain. All of the three samples were also measured by much shorter pulse with 20fs duration and 40nm bandwidth around 400nm. The measured signal rises with about 280 fs reflecting the iron displacement in the heme [3]. From these experimental results, we have proposed the simple kinetic model for ligand-heme interaction of eNOS oxygenanse domain.

Reference
[1] W. Belliston-Bittner et al., J. Am. Chem. Soc., 127, 15907-15915 (2005)
[2] I. Mikula et al, Biochem. J. 418, 673-682 (2009)
[3] J.L. Martin et al, The EMBO Journal, 2, 1815-1819 (1983)


Keywords: Ultrafast dynamics, eNOS, heme, ligand