Dapeng Sun

Involvement of a Putative [Fe-S]-cluster-binding Protein in the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestγ-subunit of theαβγ heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for theα- andγ-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The α- and β-subunits were both detected in the periplasm of the mutant strain, whereas the γ-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the γ-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of γ-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.

Lysozyme-Osmotic Shock Methods for Localization of Periplasmic Redox Proteins in Bacteria

Publisher Summary This chapter describes two different procedures that use the combination of lysozyme and osmotic shock for the fractionation of bacterial cells and selective release of periplasmic proteins. Procedure 1 has been used in our laboratory to fractionate E. coli, Paracoccus denitrificans, and Rhodobacter sphaeroides . Procedure 2 was used with Alcaligenesfaecalis , which could not be fractionated by procedure 1. The development of procedure 2 required significant modification of the generally applicable technique that was developed originally for E. coli. It is likely that these procedures will require further modificaiton for optimal effectiveness with other bacteria. As such, a general protocol is also described for optimizing conditions for the fractionation of other bacterial cells that are not efficiently fractionated by either of these two procedures.

MECHANISMS OF CATALYSIS AND ELECTRON TRANSFER BY TRYPTOPHAN TRYPTOPHYLQUINONE ENZYMES

This review covers experimental works which have been carried out on the properties of the tryptophan tryptophylquinone (TTQ) cofactor and the TTQ-containing enzyme, methylamine dehydrogenase (MADH). The kinetic mechanism of MADH catalysed reactions, factors that determine the substrate specificity of MADH and the chemical reaction mechanism of MADH are discussed in detail. Electron transfer theory and kinetic models of interprotein electron transfer are discussed. Studies of electron transfer reactions in the MADH-amicyanin-cytochrome c551i protein complex are reviewed and discussed in terms of electron transfer theory.

Redox properties of an engineered purple CuA azurin

Purple Cu(A) centers are a class of binuclear, mixed-valence copper complexes found in cytochrome c oxidase and nitrous oxide reductase. An engineered Cu(A) protein was formed by replacing a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Pseudomonas aeruginosa azurin with the corresponding portion of sequence from the Cu(A) center of cytochrome c oxidase from Paracoccus denitrificans [Proc. Natl. Acad. Sci. USA 93 (1996) 461]. Oxidation-reduction midpoint potential (E(m)) values of the Cu(A) azurin of +399+/-10 and +380+/-2mV, respectively, were determined by cyclic voltammetry and spectrochemical titration. An n value of one was obtained, indicating that the redox reaction is cycling between the mixed valence and the fully reduced states. Whereas the E(m) value of native azurin is pH dependent, the E(m) value of Cu(A) azurin is not, as expected for the Cu(A) center. Similarities and differences in the redox properties are discussed in terms of the known crystal structures of Cu(A) centers in cytochrome c oxidase and Cu(A) azurin.

Inter-subunit cross-linking of methylamine dehydrogenase by cyclopropylamine requires residue αPhe55

Cyclopropylamine is a mechanism‐based inhibitor of the quinoprotein methylamine dehydrogenase (MADH) from Paracoccus denitrificans. The resulting inactivation is accompanied by the formation of a covalent cross‐link between the α and β subunits of MADH. The results of site‐directed mutagenesis studies indicate that Phe55 on the α subunit is required for this process. No cross‐linking is seen with αF55A or αF55I MADH mutants. In contrast, with αF55E MADH cross‐linking of subunits is observed. These results suggest a novel mechanistic role for a phenylalanine residue and the possible importance of protein dynamics in this enzyme mechanism.