title: pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus.
abstract: Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.
authors.affiliation: Department of Biochemistry, Faculty of Science P. J. Safárik University, Moyzesova 11, 040 01 Kosice, Slovakia. zoltag@yahoo.co.uk
authors.forename: G
authors.initials: G
authors.lastname: Zoldák
authors.forename: A
authors.initials: A
authors.lastname: Musatov
authors.forename: M
authors.initials: M
authors.lastname: Stupák
authors.forename: M
authors.initials: M
authors.lastname: Sprinzl
authors.forename: E
authors.initials: E
authors.lastname: Sedlák
mesh_terms: D000225:Adenine / Q000737:chemistry; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002942:Circular Dichroism; D019281:Dimerization; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005415:Flavins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D013050:Spectrometry, Fluorescence / Q000379:methods; D055672:Static Electricity; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry
chemical_list: D005415:Flavins; D009097:Multienzyme Complexes; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D014364:Tryptophan; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D000225:Adenine