Attachment Of Cofactor Research Articles

HIGD2A is Required for Assembly of the COX3 Module of Human Mitochondrial Complex IV

Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. Several assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. Although in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.

COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2.

Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2.

Co-factor Insertion and Disulfide Bond Requirements for Twin-arginine Translocase-dependent Export of the Bacillus subtilis Rieske Protein QcrA

The twin-arginine translocation (Tat) pathway can transport folded and co-factor-containing cargo proteins over bacterial cytoplasmic membranes. Functional Tat machinery components, a folded state of the cargo protein and correct co-factor insertion in the cargo protein are generally considered as prerequisites for successful translocation. The present studies were aimed at a dissection of these requirements with regard to the Rieske iron-sulfur protein QcrA of Bacillus subtilis. Notably, QcrA is a component of the cytochrome bc1 complex, which is conserved from bacteria to man. Single amino acid substitutions were introduced into the Rieske domain of QcrA to prevent either co-factor binding or disulfide bond formation. Both types of mutations precluded QcrA translocation. Importantly, a proofreading hierarchy was uncovered, where a QcrA mutant defective in disulfide bonding was quickly degraded, whereas mutant QcrA proteins defective in co-factor binding accumulated in the cytoplasm and membrane. Altogether, these are the first studies on Tat-dependent protein translocation where both oxidative folding and co-factor attachment have been addressed in a single native molecule.

A conserved haem redox and trafficking pathway for cofactor attachment

A pathway for cytochrome c maturation (Ccm) in bacteria, archaea and eukaryotes (mitochondria) requires the genes encoding eight membrane proteins (CcmABCDEFGH). The CcmABCDE proteins are proposed to traffic haem to the cytochrome c synthetase (CcmF/H) for covalent attachment to cytochrome c by unknown mechanisms. For the first time, we purify pathway complexes with trapped haem to elucidate the molecular mechanisms of haem binding, trafficking and redox control. We discovered an early step in trafficking that involves oxidation of haem (to Fe(3+)), yet the final attachment requires reduced haem (Fe(2+)). Surprisingly, CcmF is a cytochrome b with a haem never before realized, and in vitro, CcmF functions as a quinol:haem oxidoreductase. Thus, this ancient pathway has conserved and orchestrated mechanisms for trafficking, storing and reducing haem, which assure its use for cytochrome c synthesis even in limiting haem (iron) environments and reducing haem in oxidizing environments.

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8alpha-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8alpha-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.

What’s in a covalent bond?

Many enzymes use one or more cofactors, such as biotin, heme, or flavin. These cofactors may be bound to the enzyme in a noncovalent or covalent manner. Although most flavoproteins contain a noncovalently bound flavin cofactor (FMN or FAD), a large number have these cofactors covalently linked to the polypeptide chain. Most covalent flavin-protein linkages involve a single cofactor attachment via a histidyl, tyrosyl, cysteinyl or threonyl linkage. However, some flavoproteins contain a flavin that is tethered to two amino acids. In the last decade, many studies have focused on elucidating the mechanism(s) of covalent flavin incorporation (flavinylation) and the possible role(s) of covalent protein-flavin bonds. These endeavors have revealed that covalent flavinylation is a post-translational and self-catalytic process. This review presents an overview of the known types of covalent flavin bonds and the proposed mechanisms and roles of covalent flavinylation.

Succinate dehydrogenase flavoprotein subunit expression in Saccharomyces cerevisiae– involvement of the mitochondrial FAD transporter, Flx1p

The mitochondrial FAD transporter, Flx1p, is a member of the mitochondrial carrier family responsible for FAD transport in Saccharomyces cerevisiae. It has also been suggested that it has a role in maintaining the normal activity of mitochondrial FAD-binding enzymes, including lipoamide dehydrogenase and succinate dehydrogenase flavoprotein subunit Sdh1p. A decrease in the amount of Sdh1p in the flx1Delta mutant strain has been determined here to be due to a post-transcriptional control that involves regulatory sequences located upstream of the SDH1 coding sequence. The SDH1 coding sequence and the regulatory sequences located downstream of the SDH1 coding region, as well as protein import and cofactor attachment, seem to be not involved in the decrease in the amount of protein.

An Essential Role for the DnaK Molecular Chaperone in Stabilizing Over-expressed Substrate Proteins of the Bacterial Twin-arginine Translocation Pathway

All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.

Covalent cofactor attachment to proteins: cytochrome c biogenesis

Haem (Fe-protoporphyrin IX) is a cofactor found in a wide variety of proteins. It confers diverse functions, including electron transfer, the binding and sensing of gases, and many types of catalysis. The majority of cofactors are non-covalently attached to proteins. There are, however, some proteins in which the cofactor binds covalently and one of the major protein classes characterized by covalent cofactor attachment is the c-type cytochromes. The characteristic haem-binding mode of c-type cytochromes requires the formation of two covalent bonds between two cysteine residues in the protein and the two vinyl groups of haem. Haem attachment is a complex post-translational process that, in bacteria such as Escherichia coli, occurs in the periplasmic space and involves the participation of many proteins. Unexpectedly, it has been found that the haem chaperone CcmE (cytochrome c maturation), which is an essential intermediate in the process, also binds haem covalently before transferring the haem to apocytochromes. A single covalent bond is involved and occurs between a haem vinyl group and a histidine residue of CcmE. Several in vitro and in vivo studies have provided insight into the function of this protein and into the overall process of cytochrome c biogenesis.

Immobilized cofactor derivatives for kinetic-based enzyme capture strategies: direct coupling of NAD(P)+

This study reevaluates the potential for direct coupling of NAD(P)+ to a carboxylate-terminating spacer arm using carbodiimide-promoted coupling in an attempt to develop a greatly simplified synthetic method for cofactor immobilization that would support the more widespread adoption of kinetic-based enzyme capture (KBEC) strategies for protein purification applications and protein-detecting arrays/proteomic studies. Direct coupling of NAD+ to epoxy (1,4-butanediol diglycidyl ether)-activated Sepharose is also described. Depending on the synthetic method used, the position of attachment of cofactor is concluded to be primarily through the pyrophosphate or ribosyl hydroxyl groups. Total substitution levels varied from 0.5 to 2μmol/g wet weight with 28–67% accessibility. Model bioaffinity chromatographic studies employing KBEC strategies are reported for bovine heart l-lactate dehydrogenase, yeast alcohol dehydrogenase, l-phenylalanine dehydrogenase from Sporosarcina, glutamate dehydrogenase (GDH) from Candida utilis, and GDH from bovine liver. The NAD+ derivative prepared using epoxy-activated Sepharose shows most potential for further development based on total substitution levels, the apparent absence of nonbiospecific interference, reversible biospecific adsorption of some of the test enzymes using soluble KBEC/stripping ligand tactics, and the relative simplicity of the synthetic method.

The design of protein-based catalysts using semisynthetic methods

The combination of site-directed mutagenesis and chemical modification has resulted in the preparation of protein conjugates with new and useful properties. Proteins modified with metal-chelating groups are proving useful for mapping tertiary and quaternary interactions using the technique of affinity cleavage. The attachment of cofactors, including pyridoxal and pyridoxamine, has resulted in the preparation of semisynthetic transaminases that display enzyme-like properties, including enantioselectivity, substrate specificity and reaction-rate acceleration.

Covalent Attachment of FAD to the Yeast Succinate Dehydrogenase Flavoprotein Requires Import into Mitochondria, Presequence Removal, and Folding

Succinate dehydrogenase (EC 1.3.99.1) in the yeast Saccharomyces cerevisiae is a mitochondrial respiratory chain enzyme that utilizes the cofactor, FAD, to catalyze the oxidation of succinate and the reduction of ubiqinone. The succinate dehydrogenase enzyme is a heterotetramer composed of a flavoprotein, an iron-sulfur protein, and two hydrophobic subunits. The FAD is covalently attached to a histidine residue near the amino terminus of the flavoprotein. In this study, we have investigated the attachment of the FAD cofactor with the use of an antiserum that specifically recognizes FAD and hence, can discriminate between apo- and holoflavoproteins. Cofactor attachment, both in vivo and in vitro, occurs within the mitochondrial matrix once the presequence has been cleaved. FAD attachment is stimulated by, but not dependent upon, the presence of the iron-sulfur subunit and citric acid cycle intermediates such as succinate, malate, or fumarate. Furthermore, this modification does not occur with C-terminally truncated flavoprotein subunits that are fully competent for import. Taken together, these data suggest that cofactor addition occurs to an imported protein that has folded sufficiently to recognize both FAD and its substrate.

Evidence for conformational changes in Escherichia coli porphobilinogen deaminase during stepwise pyrrole chain elongation monitored by increased reactivity of cysteine-134 to alkylation by N-ethylmaleimide.

Porphobilinogen deaminase from Escherichia coli becomes progressively more susceptible to inactivation by the thiophilic reagent N-ethylmaleimide (NEM) as the catalytic cycle proceeds through the enzyme-intermediate complexes ES, ES2, ES3, and ES4. Site-directed mutagenesis of potentially reactive cysteines has been used to identify cysteine-134 as the key residue that becomes modified by the reagent and leads to inactivation. Since cysteine-134 is buried at the interface between domains 2 and 3 of the E. coli deaminase molecule, the observations suggest that a stepwise conformational change occurs between these domains during each stage of tetrapyrrole assembly. Interestingly, mutation of the invariant active-site cysteine-242 to serine leads to an enzyme with up to a third of the catalytic activity found in the wild-type enzyme. Electrospray mass spectrometry indicates that serine can substitute for cysteine as the dipyrromethane cofactor attachment site.

Cysteine to serine replacements in 6-hydroxy-D-nicotine oxidase. Consequences for enzyme activity, cofactor incorporation, and formation of high molecular weight protein complexes with molecular chaperones (GroEL)

6-Hydroxy-D-nicotine oxidase, an enzyme with FAD covalently attached to the protein, contains 6 cysteine residues in positions 45 (Cys1), 59 (Cys2), 136 (Cys3), 173 (Cys4), 260 (Cys5) and 433 (Cys6). Cys2, 3, 5, and 6 were replaced with serine by site-directed mutagenesis. The effects of these exchanges on enzyme activity, the autocatalytic incorporation of the cofactor, and the interaction of the mutant proteins with molecular chaperones were analyzed. The flavinylation of 6-hydroxy-D-nicotine oxidase is dependent on the presence of allosteric effectors, e.g. glycerol 3-phosphate or other phosphorylated tricarbon compounds. Replacement of Cys2 or Cys5 abolished this dependence. Covalent incorporation of FAD was reduced to an undetectable level in the Cys3 and Cys5 mutants. Replacement of Cys6 by Ser had no significant effect on enzyme activity and cofactor attachment. Deletion of two amino acids, Phe and Arg, situated 12 and 11 amino acid residues, respectively, from the carboxyl terminus of the protein, resulted in an inactive enzyme with no covalently bound FAD. This result indicates that almost the entire protein chain has to be synthesized before the cofactor can be incorporated, making a cotranslational flavinylation step rather unlikely. The distribution of the 6-hydroxy-D-nicotine oxidase polypeptide between the high molecular weight complexes and the free soluble form was analyzed by gel filtration on Sephacryl S-200. The wild-type holoenzyme as well as the wild-type apoenzyme were recovered in the eluent fraction of the column while the mutant proteins were retained in high molecular weight complexes, predominantly in those associated with GroEL, as revealed by immunoprecipitation. The extent of complex formation with this molecular chaperone depended on the position of the mutated Cys residue within the protein. Complex formation was highest with protein from the mutants Cys2 and Cys3, less with the Cys5, and absent with the Cys6 mutant protein. Thus, alterations in the amino-terminal part of the 6-hydroxy-D-nicotine oxidase appear more important for the interaction with molecular chaperones than alterations situated in the carboxyl-terminal part of the protein.