2BC1

Structural Analysis of Streptococcus pyogenes NADH oxidase: C44S Nox

Summary for 2BC1

• Entry DOI: 10.2210/pdb2bc1/pdb
• Related: 1JOA 2bc0 2bcp
• Descriptor: NADH Oxidase, FLAVIN-ADENINE DINUCLEOTIDE (3 entities in total)
• Functional Keywords: flavoprotein, nadh oxidase, pyridine nucleotide disulfide oxidoreductase, c(4a)-peroxyflavin, conformational dynamics, cysteine oxidation, sulfinic acid, oxidoreductase
• Biological source: Streptococcus pyogenes
• Total number of polymer chains: 2
• Total formula weight: 108459.71

Authors

Boles, W.H.,Mallett, T.C. (deposition date: 2005-10-18, release date: 2006-10-31, Last modification date: 2023-08-23)

Primary citation

Wallen, J.R.,Mallett, T.C.,Okuno, T.,Parsonage, D.,Sakai, H.,Tsukihara, T.,Claiborne, A.
Structural Analysis of Streptococcus pyogenes NADH Oxidase: Conformational Dynamics Involved in Formation of the C(4a)-Peroxyflavin Intermediate.
Biochemistry, 54:6815-6829, 2015

PubMed Abstract: 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.

PubMed: 26506002
DOI: 10.1021/acs.biochem.5b00676

Experimental method

X-RAY DIFFRACTION (2.15 Å)