Structural Analysis of Streptococcus pyogenes NADH Oxidase: Conformational Dynamics Involved in Formation of the C(4a)-Peroxyflavin Intermediate.
In probing the oxygen reactivity of an Enterococcus faecalis NADH oxidase (Nox; O2 → 2H2O) C42S mutant lacking the Cys42-sulfenic acid (Cys42-SOH) redox center, we provided direct evidence of a C(4a)-peroxyflavin intermediate in the oxidative half-reaction and also described a conformational or chemical change that is rate-limiting for full reoxidation of the homodimer. In this work, the Nox from Streptococcus pyogenes (SpyNox) has been expressed and crystallized, and the overoxidized wild-type [Cys44-SOH → Cys44-sulfinic acid (Cys44-SO2H)] and C44S mutant enzyme structures have been refined at 2.0 and 2.15 Å, respectively. We show that azide binds to the two-electron reduced wild-type (EH2) enzyme and to the mutant enzyme in solution, but with a significantly higher affinity for the mutant protein. The spectral course of the titration with the SpyNox EH2 form clearly indicates progressive displacement of the Cys44-S(-) → FAD charge-transfer interaction. An azide soak with C44S Nox crystals led to the structure of the complex, as refined at 2.10 Å. The active-site N3(-) ligand is proximal to the Ser44 and His11 side chains, and a significant shift in the Ser44 side chain also appears. This provides an attractive explanation for the azide-induced loss of charge-transfer absorbance seen with the wild-type EH2 form and also permits accommodation of a C(4a)-peroxyflavin structural model. The conformation of Ser44 and the associated helical element, and the resulting steric accommodation, appear to be linked to the conformational change described in the E. faecalis C42S Nox oxidative half-reaction.
Department of Chemistry and Physics, Western Carolina University , Cullowhee, North Carolina 28723, United States.
Jamie R
JR
Wallen
Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina 27157, United States.
T Conn
TC
Mallett
Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina 27157, United States.
Takashi
T
Okuno
Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina 27157, United States.
Derek
D
Parsonage
Institute for Protein Research, Osaka University , Suita, Osaka 565-0871, Japan.
Hiroaki
H
Sakai
Institute for Protein Research, Osaka University , Suita, Osaka 565-0871, Japan.
Tomitake
T
Tsukihara
Department of Biochemistry, Wake Forest School of Medicine , Winston-Salem, North Carolina 27157, United States.
Al
A
Claiborne
D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D013293:Enterococcus faecalis / Q000201:enzymology; D005415:Flavins / Q000737:chemistry*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013297:Streptococcus pyogenes / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship
D001386:Azides; D001426:Bacterial Proteins; D005415:Flavins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; D010544:Peroxidases; C021845:NAD+ peroxidase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D003545:Cysteine