Dissociation and unfolding of inducible nitric oxide synthase oxygenase domain identifies structural role of tetrahydrobiopterin in modulating the heme environment.
The oxygenase domain of the inducible nitric oxide synthase, Delta65 iNOSox is a dimer that binds heme, L-Arginine (L-Arg), and tetrahydrobiopterin (H(4)B) and is the site for NO synthesis. The role of H(4)B 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 (H(4)B) with the bacterially expressed Delta65 iNOSox protein. Transverse urea gradient gel electrophoresis studies indicated the presence of different conformers in the cofactor-incubated and cofactor-free Delta65 iNOSox protein. Dynamic Light Scattering (DLS) studies of cofactor-incubated and cofactor-free Delta65 iNOSox protein also showed two distinct populations of two different diameter ranges. Cofactor tetrahydrobiopterin (H(4)B) 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 H(4)B is essential in modulating the iNOS heme environment and the protein environment in the dimeric iNOS oxygenase domain.
Sengupta
Rajib
R
Department of Biochemistry, University College of Science, Calcutta University, 35 Ballygunge Circular Road, Kolkata, 700 019, India.
Sahoo
Rupam
R
Ray
Sougata Sinha
SS
Dutta
Tanmay
T
Dasgupta
Anjan
A
Ghosh
Sanjay
S
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D007202:Indicators and Reagents; D008027:Light; D051379:Mice; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013056:Spectrophotometry, Ultraviolet; D014508:Urea
D007202:Indicators and Reagents; D011994:Recombinant Proteins; D001708:Biopterins; D006418:Heme; D014508:Urea; D052247:Nitric Oxide Synthase Type II; C003402:sapropterin