ABSTRACT
Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species). Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr), the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i) the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols), but not by ß-mercaptoethanol or GSH (monothiols), even in large excess; (ii) MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH) over hydrogen peroxide; (iii) MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13)×108M-1s-1). Both Cys87 and Cys154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pKa value of the Cysp residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria.
Subject(s)
Ascomycota/enzymology , Eukaryota/metabolism , Peroxidases/genetics , Peroxidases/metabolism , Amino Acid Sequence , Animals , Ascomycota/chemistry , Ascomycota/genetics , Cysteine/genetics , Eukaryota/genetics , Evolution, Molecular , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Multigene Family , Musa/microbiology , Peroxidases/chemistry , Phylogeny , Sequence Homology, Amino AcidABSTRACT
We have studied the effect of transient vibrational inversion of population in trans-ß-apo-8(')-carotenal on the time-resolved femtosecond stimulated Raman scattering (TR-FSRS) signal. The experimental data are interpreted by applying a quantum mechanical approach, using the formalism of projection operators for constructing the theoretical model of TR-FSRS. Within this theoretical frame we explain the presence of transient Raman losses on the Stokes side of the TR-FSRS spectrum as the effect of vibrational inversion of population. In view of the obtained experimental and theoretical results, we conclude that the excited S2 electronic level of trans-ß-apo-8(')-carotenal relaxes towards the S0 ground state through a set of four vibrational sublevels of S1 state.
