L. David Arscott

Methodology employed for anaerobic spectrophotometric titrations and for computer-assisted data analysis.

Publisher Summary This chapter describes the methodology employed for anaerobic spectrophotometric titrations and for computer-assisted data analysis. Many proteins that catalyze oxidoreductions have chromophoric prosthetic groups that permit the state of reduction of the protein to be monitored spectrophotometrically. The comparison of spectral data generated in the chemical manipulations of flavoproteins (or any solution with visible or ultraviolet absorbance) has been facilitated by interfacing a recording spectrophotometer with a minicomputer. Spectra can be stored on magnetic tape in a standard format and can be recalled for comparisons, mathematical manipulations, or hard-copy output. Spectra are visualized on the display terminal for comparison or manipulation, and hard-copy is made on the X-Y recorder. The procedures used for preparation of the anaerobic solutions for titration and the subsequent analysis of the data are illustrated in the chapter by describing in detail the protocol for the measurement of proton release associated with two electron reduction of lipoamide dehydrogenase by dihydrolipoamide. In these experiments, the enzyme is dissolved in an unbuffered solution containing phenol red as a pH indicator and is then reduced with dihydrolipoamide under anaerobic conditions.

The Mechanism of High Mr Thioredoxin Reductase from Drosophila melanogaster

Drosophila melanogaster thioredoxin reductase-1 (DmTrxR-1) is a key flavoenzyme in dipteran insects, where it substitutes for glutathione reductase. DmTrxR-1 belongs to the family of dimeric, high Mr thioredoxin reductases, which catalyze reduction of thioredoxin by NADPH. Thioredoxin reductase has an N-terminal redox-active disulfide (Cys57–Cys62) adjacent to the flavin and a redox-active C-terminal cysteine pair (Cys489′–Cys490′ in the other subunit) that transfer electrons from Cys57–Cys62 to the substrate thioredoxin. Cys489′–Cys490′ functions similarly to Cys495–Sec496 (Sec = selenocysteine) and Cys535-XXXX-Cys540 in human and parasite Plasmodium falciparum enzymes, but a catalytic redox center formed by adjacent Cys residues, as observed in DmTrxR-1, is unprecedented. Our data show, for the first time in a high Mr TrxR, that DmTrxR-1 oscillates between the 2-electron reduced state, EH2, and the 4-electron state, EH4, in catalysis, after the initial priming reduction of the oxidized enzyme (Eox) to EH2. The reductive half-reaction consumes 2 eq of NADPH in two observable steps to produce EH4. The first equivalent yields a FADH–-NADP+ charge-transfer complex that reduces the adjacent disulfide to form a thiolate-flavin charge-transfer complex. EH4 reacts with thioredoxin rapidly to produce EH2. In contrast, Eox formation is slow and incomplete; thus, EH2 of wild-type cannot reduce thioredoxin at catalytically competent rates. Mutants lacking the C-terminal redox center, C489S, C490S, and C489S/C490S, are incapable of reducing thioredoxin and can only be reduced to EH2 forms. Additional data suggest that Cys57 attacks Cys490′ in the interchange reaction between the N-terminal dithiol and the C-terminal disulfide.

Isolation, characterization and partial sequencing of cystine and thiol peptides of pig heart lipoamide dehydrogenase.

Abstract Pig heart lipoamide dehydrogenase (EC 1.6.4.3) contains ten half-cystines (as cysteic acid) per mole of enzyme bound FAD. Two of these are linked in an intrachain cystine which acts in concert with the flavin during catalysis. A peptic peptide containing this active center disulfide has been isolated and shown to have the sequence: Glu-Thr-Leu-Gly-Gly-Thr-Cys-Leu-Asn-Val-Gly-Cys-Ile-Pro-Ser (Lys, Ala, Leu). The enzyme also contains seven titratable thiols when either 5,5′-dithiobis (2-nitrobenzoic acid) or iodoacetate is used as titrant. Tryptic peptides containing alkylated thiols have been isolated and characterized by amino acid composition and by their positions in two-dimensional chromatography-electrophoresis. On the basis of map position and composition, the peptides containing thiols can be distinguished from one another. The results are compared with recent data of Brown and Perham (Brown, J. P. and Perham, R. N. (1974) Biochem. J. 138, 505–512) on the compositions and partial sequences of tryptic chymotryptic peptides containing half-cystines. The combined data associate nine of the ten half-cystines with unique compositions.

Properties of Lipoamide Dehydrogenase and Thioredoxin Reductase from Escherichia coli Altered by Site‐Directed Mutagenesis

Lipoamide dehydrogenase and thioredoxin reductase are members of the pyridine nucleotidedisulfide oxidoreductase family of flavoenzymes, which is distinguished by an oxidation-reduction-active disulfide.’ Other members of the family, glutathione reductase and mercuric reductase, are homologous with lipoamide dehydrogenase in all domains.”5 Thioredoxin reductase is homologous with the others only in its two adenosine binding regions; the remainder of the protein, including its active-site disulfide region, appears to have evolved convergently.6 Catalysis takes place in two half-reactions, as shown in FIGURE I for lipoamide dehydrogenase.’” In the first, dithiol-disulfide interchange effects reduction of the oxidized enzyme (E) to the 2-electron reduced form of the enzyme (EH,); and in the second, the reoxidation of EH, to E, electrons pass very rapidly via the FAD to NAD’. The distinct roles of the two nascent thiols of EH, have been demonstrated.’”” The thiol nearer the amino terminus reacts almost exclusively with iodoacetamide, and it is this thiol that interchanges with the dithiol substrate; the sulfur nearer the carboxyl terminus interacts with the FAD. Similar results are seen with glutathione reductase” and mercuric reductase.” The assignment of roles to the two nascent thiols in

Identification of acid-base catalytic residues of high-MR thioredoxin reductase from plasmodium falciparum

High-Mr thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acid-base catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.