We heterologously overproduced a hyperthermostable archaeal low potential (E m ؍ ؊62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys 42 and Cys 61 ) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Riesketype ferredoxin appear to be inequivalent as indicated by much higher stability of the His 64 Proteins containing Rieske-type [2Fe-2S] clusters are widespread in nature from hyperthermophilic Archaea and Bacteria to Eukarya and play critical electron transfer roles in various pathways such as aerobic respiration, photosynthesis, and biodegradation of various alkene and aromatic compounds (1-4). In contrast to regular plant-and vertebrate-type ferredoxins having complete cysteinyl ligations, the Rieske-type cluster has an asymmetric iron-sulfur core with the S ␥ atom of each of the two cysteine residues coordinated to one iron site and the N ␦ atom of each of the two histidine residues coordinated to the other iron site. This asymmetric ligation results in some unique redox and spectroscopic properties (for reviews, see Refs. 1 and 3-5). This cluster coordination was firmly established by recent x-ray crystal structures of several different Rieske-type protein domains (6 -11).Two different types of Rieske clusters are observed in proteins. One type displays higher reduction potentials (E m ) 1 of approximately ϩ150 to ϩ490 mV and occurs in proton-translocating respiratory complexes (cytochrome bc 1 /b 6 f complexes and their archaeal homologs without c-type cytochromes), being involved in not only electron transfer but also substrate binding and oxidation at the quinol-oxidizing Q o site (2-5, 12-15). The other type displays lower E m values of approximately Ϫ150 to Ϫ50 mV and has been found in a diverse group of bacterial multicomponent terminal oxygenases and soluble Rieske-type ferredoxins (1, 3, 8, 9, 16 -27). However, none of the latter class has been characterized in detail from any archaeal species.We recently found that the genomic DNA sequence of the thermoacidophilic archaeon Sulfolobus solfataricus strain P-1 (DSM 1616T) encodes an archaeal homolog of bacterial small Rieske-type ferredoxins with no consensus disulfide signature (DDBJ accession number AB047031 (27)). This arf gene was found by homology search against the deduced amino acid sequence of Sulfolobus tokodaii sulredoxin, a water-soluble homolog of a high potential Rieske protein (E m,low pH ϳ ϩ190 mV) with a consensus disulfide linkage (DDBJ accession number AB023295) 2 (28 -30) (Fig. 1). Subsequent cloning and heterologous overexpression in Escherichia coli of this S. solfataricus arf gene encoding the archaeal Rieske-type ferredoxin (ARF) (27) have provided an opportunity to define the influence of surrounding amino acid residues on the electronic and s...
We reported previously that a single tryptophan residue, Trp32, in human Cu,Zn-superoxide dismutase is specifically modified by peroxynitrite-CO2 [Yamakura et al. (2001) Biochim. Biophys. Acta 1548, 38-46]. In this study, we modified Cu,Zn-superoxide dismutase by using a combination of myeloperoxidase, hydrogen peroxide, and nitrite. The modified enzyme showed no loss of copper and zinc, and 15% less enzymatic activity. Trp32 was the only significant amino acid lost. After trypsin digestion of the modified SOD with peroxynitrite-CO2 and the myeloperoxidase system, six newly appearing peptides containing tryptophan derivatives were observed on microLC-ESI-Q-TOF mass analyses and HPLC with a photodiode-array detector. The derivatives of the tryptophan residue exhibiting mass increases of 4, 16 (2 peaks), 32, 45 (major), and 45 Da (minor) were identified as kynurenine, oxindole-3-alanine and its derivatives, dihydroxytryptophan, 6-nitrotryptophan and 5-nitrotryptophan, respectively. We further identified 6-nitrotryptophan from the 1H-NMR spectrum for the pronase-digested product and calculated the yield of 6-nitrotryptophan as being about 30% for each of the modification methods. The tryptophan residue in the modified human Cu,Zn-superoxide dismutase gave the same spectra for the products including 6-nitrotryptophan as the major nitrated product with the two different modification systems.
The pH-dependent resonance Raman (RR) spectral changes of the cytochrome bc1-associated, high-potential Rieske proteins have frequently been invoked to explain the redox-linked ionization behavior. We report herein RR spectral data of archaeal and bacterial Rieske proteins that directly demonstrate the pH-dependent changes near and above pKa,ox2, but not around pKa,ox1, of the visible circular dichroism (CD) transitions. The RR spectral changes are attributed to modification of the immediate [2Fe-2S] cluster environment due to deprotonation of some exchangeable amide groups in the polypeptide backbone, rather than previously assumed simple changes of the Fe-Nimid stretching vibrations.
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