Role of methionine 56 in the control of the oxidation-reduction potentials of the Clostridium beijerinckii flavodoxin: effects of substitutions by aliphatic amino acids and evidence for a role of sulfur-flavin interactions.
Flavodoxins are small electron transferases that participate in low-potential electron transfer pathways. The flavodoxin protein is able to separate the two redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor through the differential thermodynamic stabilization or destabilization of each of its redox states. In the flavodoxin from Clostridium beijerinckii, the sulfur atom of methionine 56 is in direct contact with the re or inner face of the isoalloxazine ring of the FMN cofactor. In this study, evidence was sought for a possible role for sulfur-aromatic (flavin) interactions in the regulation of one-electron reduction potentials in flavoproteins. Met56 was systematically replaced with all the naturally occurring aliphatic amino acids by site-directed mutagenesis. Replacement of Met56 with alanine or glycine increased the midpoint potentials at pH 7 for the oxidized-semiquinone couple by up to 20 mV compared to that of the wild type, while replacement by the longer chain aliphatic residues decreased the midpoint potential by >30 mV. The midpoint potential for the semiquinone-hydroquinone couple was less negative than that for the wild type for all the mutants, increasing by as much as 90 mV for the M56I mutant. For the M56A mutant, the loss of approximately 0.5 kcal/mol in the binding energy for oxidized FMN and an increase of 1. 6 kcal/mol for the flavin hydroquinone, relative to that of the wild type, are responsible for the observed changes in the midpoint potentials. The stability of the semiquinone complex of this mutant was not affected. The one-election reduction potentials for the M56L, M56I, and M56V mutants are also influenced by the differential stabilization of the three redox states; however, the semiquinone complex was significantly less stable in these proteins. These differences are likely the consequence of the introduction of additional steric factors and an apparent structural preference for a smaller or more flexible side chain at this position in the semiquinone complex. While the other factors may contribute, it is argued that the results obtained for the entire group of mutants are consistent with the elimination of important sulfur-flavin interactions that contribute in part to the stabilization of the oxidized and destabilization of the hydroquinone states of the cofactor in this flavodoxin. The results of this study also demonstrate unequivocally the functional importance of this methionine residue and that it is unique among the aliphatic amino acids in its capacity to generate the physiologically relevant low reduction potential exhibited by the C. beijerinckii flavodoxin.
Druhan
L J
LJ
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Swenson
R P
RP
D019943:Amino Acid Substitution / Q000235:genetics*; D003013:Clostridium / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D005418:Flavodoxin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013455:Sulfur / Q000378:metabolism*
D005415:Flavins; D005418:Flavodoxin; D011994:Recombinant Proteins; D013455:Sulfur; D005486:Flavin Mononucleotide; D008715:Methionine