Modulation of the Redox Potentials of FMN in Desulfovibrio vulgaris Flavodoxin: Thermodynamic Properties and Crystal Structures of Glycine-61 Mutants

详细信息    查看全文

• 作者：Paul A. O'Farrell ; Martin A. Walsh ; Andrew A. McCarthy ; Timothy M. Higgins ; Gerrit Voordouw ; and Stephen G. Mayhew
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：June 9, 1998
• 年：1998
• 卷：37
• 期：23
• 页码：8405 - 8416
• 全文大小：174K
• 年卷期：v.37,no.23(June 9, 1998)
• ISSN：1520-4995

文摘

Mutants of the electron-transfer protein flavodoxin fromDesulfovibrio vulgaris were madeby site-directed mutagenesis to investigate the role of glycine-61 instabilizing the semiquinone of FMNby the protein and in controlling the flavin redox potentials. Thespectroscopic properties, oxidation-reduction potentials, and flavin-binding properties of the mutantproteins, G61A/N/V and L, were comparedwith those of wild-type flavodoxin. The affinities of all of themutant apoproteins for FMN and riboflavinwere less than that of the wild-type apoprotein, and the redoxpotentials of the two 1-electron steps in thereduction of the complex with FMN were also affected by the mutations.Values for the dissociationconstants of the complexes of the apoprotein with the semiquinone andhydroquinone forms of FMNwere calculated from the redox potentials and the dissociation constantof the oxidized complex and usedto derive the free energies of binding of the FMN in its threeoxidation states. These showed that thesemiquinone is destabilized in all of the mutants, and that the extentof destabilization tends to increasewith increasing bulkiness of the side chain at residue 61. It isconcluded that the hydrogen bond betweenthe carbonyl of glycine-61 and N(5)H of FMN semiquinone in wild-typeflavodoxin is either absent orseverely impaired in the mutants. X-ray crystal structure analysisof the oxidized forms of the four mutantproteins shows that the protein loop that contains residue 61 is movedaway from the flavin by 5-6 Å.The hydrogen bond formed between the backbone nitrogen ofaspartate-62 and O(4) of the dimethylisoalloxazine of the flavin in wild-type flavodoxin is absent in themutants. Reliable structural informationwas not obtained for the reduced forms of the mutant proteins, but ifthe mutants change conformationwhen the flavin is reduced to the semiquinone, to facilitate hydrogenbonding between N(5)H and thecarbonyl of residue 61, then the change must be different from thatknown to occur in wild-type flavodoxin.