S-Adenosyl-l-methionine Enzyme MiaB Sulfur-donating Radical Cluster Carrier Protein with the Sulfur Monothiol Glutaredoxin and an Iron Physical and Functional Interactions of a (original) (raw)
Proceedings of the National Academy of Sciences, 2008
Ribosomal protein S12 undergoes a unique posttranslational modification, methylthiolation of residue D88, in Escherichia coli and several other bacteria. Using mass spectrometry, we have identified the enzyme responsible for this modification in E. coli, the yliG gene product. This enzyme, which we propose be called RimO, is a radical-S-adenosylmethionine protein that bears strong sequence similarity to MiaB, which methylthiolates tRNA. We show that RimO and MiaB represent two of four subgroups of a larger, ancient family of likely methylthiotransferases, the other two of which are typified by Bacillus subtilis YqeV and Methanococcus jannaschii Mj0867, and we predict that RimO is unique among these subgroups in its modification of protein as opposed to tRNA. Despite this, RimO has not significantly diverged from the other three subgroups at the sequence level even within the C-terminal TRAM domain, which in the methyltransferase RumA is known to bind the RNA substrate and which we presume to be responsible for substrate binding and recognition in all four subgroups of methylthiotransferases. To our knowledge, RimO and MiaB represent the most extreme known case of resemblance between enzymes modifying protein and nucleic acid. The initial results presented here constitute a bioinformatics-driven prediction with preliminary experimental validation that should serve as the starting point for several interesting lines of further inquiry. methylthiotransferase ͉ posttranslational modification ͉ radical-SAM protein Author contributions: B.P.A., L.S., E.A.R., S.K., and R.J.R. designed research; B.P.A., L.S., and J.S.B. performed research; B.P.A. and L.S. analyzed data; and B.P.A. wrote the paper.
Biochemistry, 2011
Monothiol glutaredoxins (mono-Grx) represent a highly evolutionarily conserved class of proteins present in organisms ranging from prokaryotes to humans. Mono-Grxs have been implicated in iron sulfur (FeS) cluster biosynthesis as potential scaffold proteins and in iron homeostasis via an FeS-containing complex with Fra2p (homologue of E. coli BolA) in yeast and are linked to signal transduction in mammalian systems. However, the function of the mono-Grx in prokaryotes and the nature of an interaction with BolA-like proteins have not been established. Recent genome-wide screens for E. coli genetic interactions reported the synthetic lethality (combination of mutations leading to cell death; mutation of only one of these genes does not) of a grxD mutation when combined with strains defective in FeS cluster biosynthesis (isc operon) functions [Butland, G., et al. (2008) Nature Methods 5, 789−795]. These data connected the only E. coli mono-Grx, GrxD to a potential role in FeS cluster biosynthesis. We investigated GrxD to uncover the molecular basis of this synthetic lethality and observed that GrxD can form FeS-bound homodimeric and BolA containing heterodimeric complexes. These complexes display substantially different spectroscopic and functional properties, including the ability to act as scaffold proteins for intact FeS cluster transfer to the model [2Fe-2S] acceptor protein E. coli apo-ferredoxin (Fdx), with the homodimer being significantly more efficient. In this work, we functionally dissect the potential cellular roles of GrxD as a component of both homodimeric and heterodimeric complexes to ultimately uncover if either of these complexes performs functions linked to FeS cluster biosynthesis. G lutaredoxins are redox proteins present in both prokaryotes and eukaryotes. CGFS-type monothiol glutaredoxins (mono-Grx) have sequence homology to classical dithiol glutaredoxins but possess a CGFS active site sequence in place of the CXXC motif present in their dithiol namesakes. 1 Detailed investigation has now revealed that mono-Grxs, while structurally similar to dithiol glutaredoxin proteins, do not function biochemically as glutaredoxins in redox chemistry. 1 Instead, studies of eukaryotic systems have connected mono-Grxs to potential roles in iron−sulfur (FeS) cluster biosynthesis, iron homeostasis, and signal transduction. 2−5 The cellular role of monothiol glutaredoxins has been most intensely studied in S. cerevisiae, which encodes three CGFStype mono-Grx homologues: Grx3p, Grx4p, and Grx5p. 1 Grx5p, which is located in the mitochondrion, is proposed to directly participate with the mitochondrial FeS cluster biosynthesis system, possibly acting to accept and/or transfer an intact FeS cluster to FeS apo-proteins. 6 Grx3p and Grx4p (herein, Grx3/4p) are cytoplasmic and display partial functional redundancy. Grx3/4p have been linked to iron regulation in S. cerevisiae by binding and influencing translocation of the transcriptional activator Aft1, which regulates iron uptake. 7,8 It should be noted that Grx3/4p contains an additional thioredoxin-like (Trx) domain upstream of the Grx domain
Methionine Sulfoxide Reductase B Displays a High Level of Flexibility
Journal of Molecular Biology, 2009
Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of methionine sulfoxide back to methionine. In vivo, Msrs are essential in the protection of cells against oxidative damage to proteins and in the virulence of some bacteria. Two structurally unrelated classes of Msrs, named MsrA and MsrB, exist. MsrB are stereospecific to R epimer on the sulfur of sulfoxide. All MsrB share a common reductase step with the formation of a sulfenic acid intermediate. For the subclass of MsrB whose recycling process passes through the formation of an intradisulfide bond, the recycling reducer is thioredoxin. In the present study, X-ray structures of Neisseria meningitidis MsrB have been determined. The structures have a fold based on two β-sheets, similar to the fold already described for other MsrB, with the recycling Cys63 located in a position favorable for disulfide bond formation with the catalytic Cys117. X-ray structures of Xanthomonas campestris MsrB have also been determined. In the C117S MsrB structure with a bound substrate, the recycling Cys31 is far from Ser117, with Trp65 being essential in the reductase step located in between. This positioning prevents the formation of the Cys31-Cys117 disulfide bond. In the oxidized structure, a drastic conformational reorganization of the two β-sheets due to withdrawal of the Trp65 region from the active site, which remains compatible with an efficient thioredoxin-recycling process, is observed. The results highlight the remarkable structural malleability of the MsrB fold.
Biochemistry, 2019
The cytosolic iron sulfur cluster assembly (CIA) scaffold, comprising Nbp35 and Cfd1 in yeast, assembles iron sulfur (FeS) clusters destined for cytosolic and nuclear enzymes. ATP hydrolysis by the CIA scaffold plays an essential but poorly understood role in cluster biogenesis. Here we find that mutation of conserved residues in the four motifs comprising the ATPase site of Nbp35 diminished the scaffold's ability to both assemble and transfer its FeS cluster in vivo. The mutants fall into four phenotypic classes which can be understood by how each set of mutations affect ATP binding and hydrolysis. In vitro studies additionally revealed that occupancy of the bridging FeS cluster binding site decreases the scaffold's affinity for nucleotide. Based on our findings, we propose that nucleotide binding and hydrolysis by the CIA scaffold drives a series of protein conformational changes that regulate association with other proteins in the
Identification and characterization of mitochondrial Mia40 as an iron–sulfur protein
Biochemical Journal, 2013
Mia40 is a highly conserved mitochondrial protein that plays an essential role in the import and oxidative folding of many proteins of the mitochondrial intermembrane space. Mia40 uses its redox active CPC motif to shuttle disulfides between its client proteins (newly imported proteins) and the thiol oxidase Erv1. As a thiol oxidoreductase, no cofactor was found in Mia40, nor is a cofactor required for this function. In the present study we, for the first time based on both in vitro and in vivo studies, show that yeast Mia40 can exist as an Fe-S (iron-sulfur) protein as well. We show that Mia40 binds a [2Fe-2S] cluster in a dimer form with the cluster co-ordinated by the cysteine residues of the CPC motifs. The biological relevance of the cofactor binding was confirmed in vivo by cysteine redox state and iron uptake analyses, which showed that a significant amount of cellular Mia40 binds iron in vivo. Furthermore, our oxygen consumption results suggested that the Fe-S-containing Mia40 is not an electron donor for Erv1. Thus we conclude that Mia40 is a novel Fe-S protein with a new clusterbinding motif (CPC), and apart from the thiol oxidoreductase activity, Mia40 may have another important, as yet undefined, function in cells.
2016
The redox-active type 1 copper site of amicyanin is composed of a single copper ion that is coordinated by two histidines, a methionine, and a cysteine residue. This redox site has a potential of +265 mV at pH7.5. Over ten angstroms away from the copper site resides a tryptophan residue whose fluorescence is quenched by the copper. The effects of the tryptophan on the electron transfer (ET) properties were investigated by site-directed mutagenesis. Lessons learned about the hydrogen bonding network of amicyanin from the aforementioned study were attempted to be used as a model to increase the stability of another beta barrel protein, the immunoglobulin light chain variable domain (V L). In addition, amicyanin was used as an alternative redox partner with MauG. MauG is a diheme protein from the mau gene cluster that catalyzes the biogenesis of the tryptophan tryptophylquinone cofactor of methylamine dehydrogenase (MADH). The amicyanin-MauG complex was used to study the free energy dependence and impact of reorganization energy in biological electron transfer reactions. The sole tryptophan of amicyanin was converted to a tyrosine via site-directed mutagenesis. This mutation had no effect on the electron transfer parameters with its redox partners, methylamine dehydrogenase and cytochrome c-551i. However, the pKa of the pH-dependence of the redox potential of the copper site was shifted +0.5 pH units. This was a result of an additional hydrogen bond between Met51 and the copper coordinating residue His95 in the reduced form of amicyanin. This additional hydrogen bond stabilizes the reduced form. Also, the stability of the copper site and the protein overall was significantly decreased, as seen by the temperature dependence of the visible spectrum of the copper site and the circular dichroism spectrum of the protein. This destabilization is attributed to the loss of an interior, crossbarrel hydrogen bond. The V L is structurally similar to amicyanin, but it does not contain any cross-barrel hydrogen bonds. The importance of the cross-barrel hydrogen bond in stabilizing amicyanin is evident. A homologous bond in iv V L was attempted to be engineered by using site-directed mutagenesis to insert neutral residues with protonatable groups into the core of the protein. Wild-type (WT) V L was purified from the periplasm and found to be properly folded as determined by circular dichroism and size exclusion chromatography. Mutants were expressed in E. coli using the amicyanin signal sequence for periplasmic expression. Folded mutant protein could not be purified from the periplasm. When amicyanin is used in complex with MauG, it retains the pH-dependence of the redox potential of its copper site due to the looseness of the interprotein interface. The free energy of the reaction was manipulated by variation in pH from pH 5.8 to 8.0. The ET parameters are reorganization energy of 2.34 eV and an electronic coupling constant of 0.6 cm-1. P94A amicyanin has a potential that is 120 mV higher than WT amicyanin and was used to extend the range of the free energy dependence studied. The ET parameters of the reaction of WT and P94A amicyanin with MauG were within error of each other. This is significant because the ET reaction of P94A amicyanin with its natural electron acceptor was not able to be studied due to a kinetic coupling of the ET step with a nonET step. This kinetic coupling obscured the parameters of the ET step because it is not kinetically distinguishable from the ET step. A Y294H MauG mutant was also studied. This mutation replaced the axial tyrosine ligand of the sixcoordinate heme of MauG with a histidine. No reaction is observed with Y294H MauG in its native reaction. However, the high valent oxidation state of the five-coordinate heme of Y294H MauG reacts with reduced amicyanin. The ET rate was analyzed by ET theory to study the high valent heme in Y294H MauG. The reorganization energy of Y294H MauG was calculated to be nearly 20% lower as compared to the same reaction with WT MauG. These results provide insight into the obscured nature of reorganization energy of large redox cofactors in proteins, particularly heme cofactors, as well as to how the active sites of enzymes are optimized to perform long range ET vs catalysis with regard to balancing redox potential and reorganization energy. v Dedicated to my family who has always supported me through every challenge Keishla, Danica Walt, Dianne, & Matt vi ACKNOWLEDGMENTS I express my deepest gratitude and appreciation to my famous, expert advisor and mentor, Dr. Victor L. Davidson for his guidance, patience, and example. In a world of fluorescent microscopes, he has been able to show me the utility and advantage of studying basic biochemistry and enzyme kinetics. Also I thank him for emphasizing the principle of Ockham's razor and not letting me (or anyone else in the lab) get swept away by overly complicated or unprecedented ideas. I thank my committee members, Suren Tatulian, William Self, and Kyle Rohde for technical support in performing experiments and also for supporting me and my work in the Burnett School of Biomedical Sciences. I also appreciate and thank Dr. Griff Parks, Dr. Sampath Parthasarathy, and Dr. Richard Peppler for their vision, guidance, and support of our lab and department as a whole. Particular thanks go to the rest of our lab group who have helped me keep my sanity, and, at times, tolerate my insanity. Specifically to Esha Sehanobish for her camaraderie, empathy, and teamwork; Heather Williamson for being a readily accessible font of biochemistry trivia; Yu Tang for her understanding and selfless willingness to express and purify proteins and provide buffers solutions, gels, and more. She is a most valuable anchor in the lab; Moonsung Choi and Sooim Shin for welcoming me to the lab and providing expertise in designing and performing experiments. Throughout my tenure at UCF, I have also enjoyed the company and friendship of many of my fellow students, especially Jason O. Matos, Aladdin Riad, and Richard Barrett, and everyone in the Biomedical Sciences Graduate Student Association. I thank them as well for helping me maintain my sanity. Finally, I thank my love, Keishla, and our beautiful daughter, Danica. They have always been on my mind or literally looking over my shoulder, demanding hard work, precision, integrity, and excellence. They have also both had unbelievable patience and understanding with me during my studies. Gracias. vii
2016
siae glutaredoxin involved in iron-sulfur cluster (FeSC) biogenesis. Previouswork suggests thatGrx5 is involved in regulating protein cysteine glutathio-nylation, prompting several questions about the systemic role of Grx5. First, is the regulation of mixed protein-glutathione disulfide bridges in FeSC biosynthetic proteins by Grx5 sufficient to account for the observed phenotypes of the grx5 mutants? If so, doesGrx5 regulate the oxidation state ofmixed protein-glutathione disulfide bridges in FeSC bio-genesis in general? Alternatively, can the grx5 mutant phenotypes be explained if Grx5 acts on just one or a few of the FeSC biogenesis proteins? In the first part of this article, we address these questions by building and analyzing amathematical model of FeSC biosynthesis. We show that, indepen-dent of the tested parameter values, the dynamic behavior observed in cells depleted of Grx5 can only be qualitatively reproduced if Grx5 acts by regulat-ing the initial assembly of FeSC in s...
[2Fe-2S] cluster transfer in iron-sulfur protein biogenesis
Proceedings of the National Academy of Sciences of the United States of America, 2014
Monothiol glutaredoxins play a crucial role in iron-sulfur (Fe/S) protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of [2Fe-2S] clusters from scaffold proteins to target apo proteins, possibly by acting as cluster transfer proteins. The molecular basis of [2Fe-2S] cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe/S proteins and participates in cellular iron regulation. Here we show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein-protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] clus...