Exploring second coordination sphere effects in nitric oxide synthase.
Second coordination sphere (SCS) effects in proteins are modulated by active site residues and include hydrogen bonding, electrostatic/dipole interactions, steric interactions, and π-stacking of aromatic residues. In Cyt P450s, extended H-bonding networks are located around the proximal cysteinate ligand of the heme, referred to as the 'Cys pocket'. These hydrogen bonding networks are generally believed to regulate the Fe-S interaction. Previous work identified the S(Cys) → Fe σ CT transition in the high-spin (hs) ferric form of Cyt P450cam and corresponding Cys pocket mutants by low-temperature (LT) MCD spectroscopy [Biochemistry 50:1053, 2011]. In this work, we have investigated the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in the hs ferric state (with H4B and L-Arg bound) of rat neuronal nitric oxide synthase oxygenase construct (nNOSoxy) using MCD spectroscopy. For this purpose, wt enzyme and W409 mutants were investigated where the H-bonding network with the axial Cys ligand is perturbed. Overall, the results are similar to Cyt P450cam and show the intense S(Cys) → Fe σ CT band in the LT MCD spectrum at about 27,800 cm-1, indicating that this feature is a hallmark of {heme-thiolate} active sites. The discovery of this MCD feature could constitute a new approach to classify {heme-thiolate} sites in hs ferric proteins. Finally, the W409 mutants show that the hydrogen bond from this group only has a small effect on the Fe-S(Cys) bond strength, at least in the hs ferric form of the protein studied here. Low-temperature MCD spectroscopy is used to investigate the effect of the hydrogen bond from W409 to the axial Cys ligand of the heme in neuronal nitric oxide synthase. The intense S(Cys) → Fe σ-CT band is monitored to identify changes in the Fe-S(Cys) bond in wild-type protein and W409 mutants.
Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.|Department of Biophysics, University of Michigan, Ann Arbor, MI, 48109, USA.
Ashley B
AB
McQuarters
Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA.|Department of Biophysics, University of Michigan, Ann Arbor, MI, 48109, USA.
Amy L
AL
Speelman
College of Pharmacy, University of New Mexico, Albuquerque, NM, 87131, USA.
Li
L
Chen
College of Pharmacy, University of New Mexico, Albuquerque, NM, 87131, USA.
Bradley O
BO
Elmore
College of Pharmacy, University of New Mexico, Albuquerque, NM, 87131, USA.
Weihong
W
Fan
College of Pharmacy, University of New Mexico, Albuquerque, NM, 87131, USA.
Changjian
C
Feng
Department of Chemistry, University of Michigan, Ann Arbor, MI, 48109, USA. lehnertn@umich.edu.|Department of Biophysics, University of Michigan, Ann Arbor, MI, 48109, USA. lehnertn@umich.edu.
Nicolai
N
Lehnert
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain; D002942:Circular Dichroism / Q000379:methods; D056831:Coordination Complexes / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D013053:Spectrophotometry; D055672:Static Electricity; D013816:Thermodynamics
D056831:Coordination Complexes; D008024:Ligands; D009569:Nitric Oxide; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D052248:Nitric Oxide Synthase Type I; D003545:Cysteine
Cytochrome P450; Electronic Spectra; Heme; Magnetic circular dichroism; Nitric oxide synthase; Second coordination sphere effects
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