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Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment

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Abstract

The oxygenase domain of the inducible nitric oxide synthase, Δ65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H4B) and is the site for NO synthesis. The role of H4B in iNOS structure-function is complex and its exact structural role is presently unknown. The present paper provides a simple mechanistic account of interaction of the cofactor tetrahydrobiopterin (H4B) with the bacterially expressed Δ65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Δ65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Δ65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H4B) shifted one population, with higher diameter, to the lower diameter ranges indicating conformational changes. The additional role played by the cofactor is to elevate the heme retaining capacity even in presence of denaturing stress. Together, these findings confirm that the H4B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain. (Mol Cell Boichem xxx: 1–10, 2005)

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Abbreviations

NO:

nitric oxide

nNOS:

neuronal nitric oxide synthase

iNOS:

inducible nitric oxide synthase

eNOS:

endothelial nitric oxide synthase L-Arg, L-Arginine

H4B:

(6R)-5,6,7,8-tetrahydro-Lbiopterin

NADPH:

reduced β-nicotinamide adenine dinucleotide

NOS FL:

full-length NOS subunit

ox:

oxygenase subunit

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Authors and Affiliations

  1. Department of Biochemistry, University College of Science, Calcutta University, 35 Ballygunge Circular Road, Kolkata, 700 019, India

    Rajib Sengupta, Rupam Sahoo, Sougata Sinha Ray, Tanmay Dutta, Anjan Dasgupta & Sanjay Ghosh

  2. Department of Biochemistry, University College of Science, Calcutta University, 35 Ballygunge Circular Road, Kolkata, 700 019, India

    Sanjay Ghosh

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  1. Rajib Sengupta
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  2. Rupam Sahoo
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  3. Sougata Sinha Ray
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  4. Tanmay Dutta
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  5. Anjan Dasgupta
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  6. Sanjay Ghosh
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Correspondence to Sanjay Ghosh.

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Supported by Calcutta University Research Grants.

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Sengupta, R., Sahoo, R., Ray, S.S. et al. Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment. Mol Cell Biochem 284, 117–126 (2006). https://doi.org/10.1007/s11010-005-9027-0

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