The midpoint potentials for the oxidized-semiquinone couple for Gly57 mutants of the Clostridium beijerinckii flavodoxin correlate with changes in the hydrogen-bonding interaction with the proton on N(5) of the reduced flavin mononucleotide cofactor as measured by NMR chemical shift temperature dependencies.
In the Clostridium beijerinckii flavodoxin, the reduction of the flavin mononucleotide (FMN) cofactor is accompanied by a local conformation change in which the Gly57-Asp58 peptide bond "flips" from primarily the unusual cis O-down conformation in the oxidized state to the trans O-up conformation such that a new hydrogen bond can be formed between the carbonyl group of Gly57 and the proton on N(5) of the neutral FMN semiquinone radical [Ludwig, M. L., Pattridge, K. A., Metzger, A. L., Dixon, M. M., Eren, M., Feng, Y., and Swenson, R. P. (1997) Biochemistry 36, 1259-1280]. This interaction is thought to contribute to the relative stabilization of the flavin semiquinone and may be at least partially responsible for the substantial separation of the midpoint potentials of the two one-electron reduction steps. Through a series of amino acid substitutions, the above cited study demonstrated the critical role of the often conserved glycine residue in this process. However, it has not been directly established experimentally as to whether these substitutions brought about the changes in the midpoint potentials by altering the strength of this hydrogen-bonding interaction as proposed. In this study, the relative strengths of the FMN N(5)H.O57 hydrogen bond in wild type and the G57A, G57N, and G57T mutants were evaluated by measuring the temperature dependency of the chemical shift for the proton on N(5) of the fully reduced cofactor by 1H-15N HSQC nuclear magnetic resonance spectroscopy. Based on the established correlation between the temperature coefficient of amide protons and the strength of hydrogen bonding in small peptides, the apparent strength of the N(5)H.O57 interaction was found to depend on the properties of the side chain at position 57. The glycine residue found in the wild-type flavodoxin appears to provide the strongest interaction while the beta-branched side chain in the G57T mutant provides the weakest. A good correlation was noted between the temperature coefficients of N(5)H and the one-electron reduction potential for the ox/sq couple as well as the binding free energy of the FMN semiquinone in this group of mutants. These results provide more direct quantitative evidence that support the previous hypothesis that this conformation change and the associated formation of the hydrogen bonding interaction with N(5)H of the reduced FMN represent an important means of stabilizing the neutral semiquinone and in modulating the oxidation-reduction potentials of the flavin cofactor in this and perhaps other flavodoxins.
Chang
F C
FC
Department of Biochemistry, Biophysics Graduate Program, The Ohio State University, Columbus 43210, USA.
Swenson
R P
RP
D019943:Amino Acid Substitution / Q000235:genetics; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016227:Benzoquinones / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D003013:Clostridium / Q000737:chemistry / Q000235:genetics; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005998:Glycine / Q000235:genetics*; D006860:Hydrogen Bonding; D006873:Hydroquinones / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011522:Protons; D013696:Temperature
D001059:Apoproteins; D016227:Benzoquinones; D005418:Flavodoxin; D006873:Hydroquinones; D009587:Nitrogen Isotopes; D011522:Protons; C026932:apoflavodoxin; C025232:semiquinone radicals; D005486:Flavin Mononucleotide; D005998:Glycine