Lipoamide Dehydrogenase from Azotobacter vinelandii: site‐directed mutagenesis of the His450‐Glu455 diad

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Abstract
Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced by other residues. His450, the active‐site base, was changed into Ser, Tyr and Phe. Pro451, in cis conformation, was changed into Ala. Glu455 was replaced with Asp and Gln. Absorption, fluorescence and CD spectroscopy of the mutated enzymes in their oxidized state (Eox) showed only minor changes with respect to the wild‐type enzyme, wherease considerable changes were observed in the spectra of the two‐electron‐reduced (EH2) species of the enzymes upon reduction by the substrate dihydrolipoamide. Differences in extent of reduction of the flavin by NADH indicate that the redox potential of the flavin is altered by the mutations. Enzyme Pro455 Ala showed the greatest deviation from wild type. The enzyme is very easily over‐reduced to the four‐electron reduced state (EH4) by dihydrolipoamide. This is probably due to a change in the backbone conformation caused by the cis‐trans conversion. From studies on the pH dependence of the thiolate charge‐transfer absorption and the relative fluorescence of EH2 of the enzymes, it is concluded that mutation of His450 results in a relatively simple and easily interpreted distribution of electronic species at the EH2 level. For all three His450‐mutated enzymes an apparent pKa1 near 5.5 is calculated that is assigned to the interchange thiol. A second apparent pKa2 is calculated of 6.9, 7.5 and 7.1 for the His450 Phe, ‐Ser and ‐Tyr enzymes, respectively, and signifies the deprotonation of the tautomeric equilibrium between the interchange and charge‐transfer thiols. The difference in apparent pK2 values between the His45‐mutated enzymes is explained by changes in micropolarity. At the EH2 level the wild‐type enzyme consists of multiple electronic forms as reported for the Escherichia coli enzyme [Wilkinson, K. D. and Williams C. H. Jr (1979) J. Biol. Chem. 254, 852–862]. Based on the results obtained with the His450‐mutated enzymes, it is concluded that the lowest pKa is associated with the interchange thiol. A model for the equilibrium species of the wild‐type enzyme at the EH2 level is presented which takes three pKa values into account. The results of the pH dependence of the electronic species at the EH2 level of Glu455‐mutated enzymes essentially follow the model proposed for the wild‐type enzyme. However mutation of Glu455 shifts the tautomeric equilibrium of EH2 in favor of the charge‐transfer species. This effect is explained by impaired ion‐pair formation between the protonated His and the interchange thiolate and a lowering of the pka of His450.