Carboxyl residues in the iron-sulfur protein are involved in the proton pumping activity of P. denitrificans bc(1) complex.

A study is presented on chemical modification of the three subunit Paracoccus denitrificans bc(1) complex. N-(Ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment caused a loss of the proton pumping activity of liposome-reconstituted bc(1) complex. A similar effect, which is referred to as the decoupling effect, resulted upon reaction of N,N'-dicyclohexylcarbodiimide (DCCD) with the complex. Direct measurement of the binding of EEDQ to the complex subunits, performed in the presence of the fluorescent hydrophobic nucleophile 4'-[(aminoacetamido)methyl]fluorescein (AMF), showed that the iron-sulfur protein (ISP) and cytochrome c(1) were labeled by EEDQ, whereas cytochrome b was not. Tryptic digestion and sequencing analysis of the fluorescent fragment of the ISP revealed this to consist of a segment with six acidic residues, among which the highly conserved aspartate 160 is present. Analogous experiments on DCCD binding showed that all the three subunits of the complex were labeled. However, DCCD concentration dependence of carboxyl residue modification in the individual subunits and of proton pumping activity showed that the decrease of the H(+)/e(-) ratio correlated only with the modification of the ISP. Tryptic digestion of labeled ISP and sequencing analysis of the fluorescent fragment gave results superimposable upon those obtained with EEDQ. Chymotryptic digestion and sequencing analysis of the single fluorescent fragment of cytochrome b showed that this fragment contained glutamate 174 and aspartate 187. We conclude that, in the P. denitrificans bc(1) complex, carboxyl residues in cytochrome b do not appear to be critically involved in the proton pump mechanism of the complex.     

Structural and functional characteristics of polypeptide subunits of the bovine heart ubiquinol--cytochrome-c reductase complex.

Structural and functional characteristics of subunits of bovine heart cytochrome-c reductase have been investigated by controlled digestion of soluble and membrane-reconstituted purified bc1 complex and direct amino acid sequencing of native and digested protein subunits. The results obtained show that the N-terminal segments of core protein II and the 14-kDa protein extend at the periphery of the complex, protruding into the inner matrix space. The Fe-S protein, located at the outer C-periphery of the complex, is shown to be anchored to other subunits of the complex by the amphipathic N-terminal region. Proteolytic cleavage of 7-11 residues from the N-terminal segment of the 14-kDa protein is apparently associated with decoupling of redox-linked proton pumping. Partial digestion of core protein II, the 6.4-kDa protein, and the C-terminal region of the 9.2-kDa protein, is without effect on the redox and proton-motive activity of the complex.     

Proton translocation by the H+-ATPase of mitochondria. Effect of modification by monofunctional reagents of thiol residues in F0 polypeptides

A study is presented on the effect of chemical modification of thiol groups on proton conduction by the H+-ATPase complex in 'inside out' submitochondrial particles, before and after removal of the F1 moiety, and by F0 liposomes. The results obtained show that modification with monofunctional reagents [N-ethylmaleimide, 2,2'-dithiobispyridine, mersalyl and N-(7-dimethylamino-4-methyl-coumarinyl)-maleimide] of thiol residues in membrane integral proteins of F0 results in inhibition of proton conduction. Comparison of the inhibitory effects with the binding of [14C]N-ethylmaleimide to the various F0 polypeptides indicates that the inhibition of proton conduction by thiol reagents was correlated with modification of the 25-kDa, 11-kDa and 9-kDa (N,N'-dicyclohexylcarbodiimide-binding protein) proteins. Involvement of the last component is supported by the observation that modification by thiol reagents depressed the binding of N,N'-dicyclo[14C]hexylcarbodiimide to the 9-kDa protein.     

Inactivation of the mitochondrial ATPase inhibitor protein by chemical modification with diethylpyrocarbonate

Modification of histidine residue(s) by diethylpyrocarbonate treatment of submitochondrial particles obtained by sonication results in inhibition of ATPase activity and stimulation of oligomycin-sensitive H+ conduction. The inhibition of the ATPase (EC 3.6.1.3) activity persisted in F1 isolated from diethylpyrocarbonate-treated submitochondrial particles, which exhibited the absorbance spectrum of modified histidine. Thus the inhibition of the ATPase activity results from histidine modification in F1 subunits. Removal of the natural inhibitor protein from submitochondrial particles resulted in stimulation of proton conduction. After removal of F1 inhibitor protein from the particles the stimulatory effect exerted by diethylpyrocarbonate treatment on proton conduction was lost. Reconstitution experiments showed that purified F1 inhibitor protein lost, after histidine modification, its capacity to inhibit the ATPase activity and proton conduction. These observations show that the stimulation of proton conduction by the ATPase complex effected by diethylpyrocarbonate treatment results from histidine modification in F1 inhibitor protein.     

Chemical modification studies of beef-heart mitochondrial b-c1 complex. Effect of modification by ethoxyformic anhydride

The effect of the histidine-modifier ethoxyformic anhydride (EFA) on the enzymatic properties of the mitochondrial b-c1 complex (ubiquinol-cytochrome c reductase) has been investigated. Chemical modification by EFA inhibited to the same extent the reductase and the proton translocating activity of the complex. In particular EFA modification of the complex resulted in: strong inhibition of the antimycin-insensitive reduction of b cytochromes; inhibition of the antimycin-promoted oxidant-induced reduction of b cytochromes and inhibition of oxidation of pre-reduced b cytochromes. Analysis of the absorbance at 238 nm, indicative of N-(ethoxyformyl)histidine derivative, of the various polypeptide subunits separated by high-pressure liquid chromatography procedure, showed that EFA modified residues in core proteins and in the low-molecular-mass proteins. Both the inhibition of the redox and the protonmotive activity of the complex and the absorbance increase at 238 nm of the core protein fraction were readily reversed by hydroxylamine, indicating that modification of histidine residue(s) in core protein(s) is critical for the activity of the complex. This was supported by the finding that modification of the reductase with EFA prevented binding of fluorescein isothiocyanate to histidine residue(s) in core protein II. EFA modification of the reductase was without effect on the binding of N-(7-dimethylamino-4-methylcoumarinyl)maleimide to the various polypeptides of the complex except for the binding to the Fe-S protein which was greatly potentiated. Thus primary chemical modification of histidine residue(s) in core protein (II) appears to cause, in turn, a conformational change in the Rieske Fe-S protein.     

Redox-linked proton translocation in the b-c1 complex from beef-heart mitochondria reconstituted into phospholipid vesicles. Studies with chemical modifiers of amino acid residues

Possible involvement of polypeptides of b-c1 complex of beef-heart mitochondria in its redox and protonmotive activity has been investigated, by means of chemical modification of amino acid residues in the soluble as well as in the phospholipid-reconstituted b-c1 complex. Treatment of the enzyme with tetranitromethane (C(NO2)4) or with ethoxyformic anhydride (EFA), that modify reversibly tyrosyl and hystidyl residues respectively, resulted in a marked inhibition of electron transport from reduced quinols to cytochrome c. This was accompanied, in b-c1 reconstituted into phospholipid vesicles, by a parallel inhibition of respiratory-linked proton translocation; the H+/e- stoichiometry remained unchanged. Treatment of b-c1 complex with DCCD, that specifically modifies carboxylic groups of glutammic or aspartic residues caused a marked depression of proton translocation in b-c1 vesicles, under conditions where the rate of electron flow in the coupled state, was enhanced. As a consequence the H+/e- stoichiometry was lowered. SDS gel electrophoresis and [14C]DCCD-labelling of the polypeptides of the b-c1 complex showed a major binding of 14C-DCCD to the 8-kDa subunit of the complex and possible cross-linking, induced by DCCD treatment, of polypeptide(s) in the 8-kDa band and the 12-kDa band, with the Fe-s protein of the complex, with the appearance of a new polypeptide band with an apparent molecular mass of about 40 kDa. Involvement of polypeptides of low molecular mass, for which no functional role was so far described, and possibly of the Fe-S protein in the redox-linked proton translocation in b-c1 complex is suggested.     

Structure-function relationships in heparin cofactor II: chemical modification of arginine and tryptophan and demonstration of a two-domain structure

Heparin cofactor II and antithrombin III are plasma proteins functionally similar in their ability to inhibit thrombin at accelerated rates in the presence of heparin. To further characterize the structural and functional properties of human heparin cofactor II as compared to antithrombin III, we studied the possible significance of arginyl and tryptophanyl residues and the changes in protein structure and activity during guanidinium chloride (GdmCl) denaturation. Both antithrombin and heparin cofactor activities of heparin cofactor II are inactivated by the arginine-specific reagent, 2,3-butanedione. Saturation kinetics are observed during modification and suggest formation of a reversible protease inhibitor-butanedione complex. Quantitation of arginyl residues following butanedione modification shows a loss of about four residues for total inactivation, one of which is essential for antithrombin activity. Arginine-modified heparin cofactor II did not bind to heparin-agarose and implies a role for the other modified arginyl residues during heparin cofactor activity. N-Bromosuccinimide oxidation (20 mol of reagent/mol of protein) of heparin cofactor II results in modification of approximately two tryptophanyl residues with no concomitant loss of heparin cofactor activity. Moreover, there is no enhancement of intrinsic protein fluorescence during heparin binding to the native inhibitor. Circular dichroism measurements show that the structural transition of heparin cofactor II during denaturation is distinctly biphasic, yielding midpoints at 0.6 and 2.6 M GdmCl. Functional protease inhibitory activities are affected to the same extent following denaturation-renaturation at various GdmCl concentrations. The results indicate that arginyl residues are critical for both antithrombin and heparin binding activities. In contrast, tryptophanyl residues are apparently not essential for heparin-dependent interactions. The results also suggest that heparin cofactor II contains two structural domains which unfold at different GdmCl concentrations.     

Iodination of ovotransferrin and its iron complex. Extent of involvement of tyrosine phenolic groups in the iron binding

Short-time iodination of metal-free ovotransferrin indicated that the tyrosine groups involved in the iron-binding activity are indistinguishable from other structural tyrosines. Modification of a minimum of 14 tyrosine residues per molecule of protein was required to achieve a complete loss of metal-binding activity. In contrast, a maximum modification of 10 tyrosine residues in iron-ovotransferrin complex could be produced with no loss of iron-binding activity. The difference in the extent of modification of tyrosines, therefore, indicated the involvement of four tyrosines in the binding of two atoms of iron. A minimal modification of histidine residues was also found, which was limited to one residue per molecule of both ovotransferrin and its iron complex. The possible participation of two tryptophan residues in the iron-binding activity is also suggested in the present study.     

The proton pump of the mitochondrial bc1 complex

The molecular mechanism of the proton pump activity by the respiratory chain bc1 complex is still unknown. This group has proposed since long time that protonation/deprotonation events in the apoproteins of the complex are cooperatively linked to the oxido-reduction reactions at the quinone catalytic centre. Protolytic residues in the apoproteins can thus provide proton transfer pathways between the bulk aqueons phases and the redox centre. A series of experiments has been carried out aimed at demonstrating a role of particular complex subunits in the pump process. In this paper recent results are reviewed which have evidenced a definite role of polypeptide carboxyl residues in the proton pump mechanism. In particular, experiments carried out with both the bovine and P. denitrificans purified enzymes have indicated a specific involvement of aspartic residue(s) in the Rieske Fe/S protein in the proton pump function.     

A histidine residue at the active site of avian liver phosphoenolpyruvate carboxykinase

The histidine-selective reagents diethylpyrocarbonate (DEPC) and dimethylpyrocarbonate were used to study active site residues of phosphoenolpyruvate carboxykinase. Both reagents show pseudo first-order inhibition of enzyme activity at 22 +/- 1 degree C with calculated second-order rate constants of 2.8 and 4.6 M-1 s-1, respectively. The inhibition appears partially reversible. Substrates affect the rate of inhibition: KHCO3 enhances the rate, Mn2+ has little effect, and phosphoenolpyruvate decreases the rate. The best protection is obtained by IDP or IDP and Mn2+. The kinetic studies show that modification of histidine is specific and leads to loss of enzymatic activity. Two histidines per enzyme are modified by DEPC, as measured by an absorption change at 240 nm, in the absence of substrate, leading to loss in activity. One histidine per molecule is modified in the presence of KHCO3, giving inactivation. Cysteine and lysine residues are not affected. A study of the inhibition rate constant as a function of pH gives a pKa of 6.7. Enzyme modified by DEPC in the absence of substrate (1% remaining activity) shows no binding of ITP or of phosphoenolpyruvate to the enzyme.Mn2+ complex as studied by proton relaxation rates. When enzyme is modified in the presence of KHCO3 (44% remaining activity), ITP and KHCO3 bind to the enzyme.Mn2+ complex similarly to the binding to native enzyme. Phosphoenolpyruvate binding to modified enzyme.Mn results in an enhancement of proton relaxation rates rather than the decrease observed with native enzyme.Mn. The CD spectra of histidine-modified enzyme show a decrease in alpha-helical and random structure with an increase in anti-parallel beta-sheet structure compared to native enzyme. These results show that avian phosphoenolpyruvate carboxykinase has 2 histidine residues which are reactive with DEPC and dimethylpyrocarbonate, and one of the 15 histidine residues in the protein is at or near the phosphoenolpyruvate binding site and is involved in catalysis.     

The role of the MoFe protein alpha-125Phe and beta-125Phe residues in Azotobacter vinelandii MoFe protein-Fe protein interaction

Site-directed mutagenesis and gene-replacement techniques were used to substitute alanine for the MoFe protein alpha- and beta-subunit phenylalanine-125 residues both separately and in combination. These residues are located on the surface of the MoFe protein near the pseudosymmetric axis of symmetry between the alpha- and beta-subunits. Altered MoFe proteins that contain an alanine substitution at only one of the respective positions exhibit proton reduction activities of about 25-50% when compared to that of the wild-type protein. The lower level of proton reduction also corresponds with decreases in the rates of MgATP hydrolysis. The MoFe protein which contains alanine substitutions in both the alpha- and beta- subunits did not exhibit any proton reduction activity or MgATP hydrolysis. Stopped flow spectrophotometry of the singly substituted MoFe proteins indicate primary electron transfer rate constants approximately an order of magnitude slower than what is observed for wild-type MoFe protein, while no primary electron transfer is observed for the doubly substituted MoFe protein. The doubly substituted MoFe protein is able to interact with the Fe protein as shown by chemical crosslinking experiments. However, this protein does not form a tight complex with the Fe protein when treated with MgADP-AlF4- or when using the altered 127delta Fe protein. Stopped flow spectrophotometry was also used to quantitate the first-order dissociation rate constants for the two component proteins. These results suggest that the 125Phe residues are involved in an early event(s) that occurs upon component protein docking and could be involved in eliciting MgATP hydrolysis.     

Palmitoylation and polymerization of hepatitis C virus NS4B protein

Hepatitis C Virus (HCV) NS4B protein induces a specialized membrane structure which may serve as the replication platform for HCV RNA replication. In the present study, we demonstrated that NS4B has lipid modifications (palmitoylation) on two cysteine residues (cysteines 257 and 261) at the C-terminal end. Site-specific mutagenesis of these cysteine residues on individual NS4B proteins and on an HCV subgenomic replicon showed that the lipid modifications, particularly of Cys261, are important for protein-protein interaction in the formation of the HCV RNA replication complex. We further demonstrated that NS4B can undergo polymerization. The main polymerization determinants were mapped in the N-terminal cytosolic domain of NS4B protein; however, the lipid modifications on the C terminus also facilitate the polymerization process. The lipid modification and the polymerization activity could be two properties of NS4B important for its induction of the specialized membrane structure involved in viral RNA replication.     

Different functions of MdtB and MdtC subunits in the heterotrimeric efflux transporter MdtB(2)C complex of Escherichia coli

In contrast to homotrimeric transporters of the RND (resistance-nodulation-division) superfamily, which often conduct efflux transport of a wide range of substrates by the functionally rotating mechanism, the mechanism utilized by the heterotrimeric members of this family, which also perform multidrug efflux, is unclear. We examined one heterotrimeric transporter, the MdtB(2)C complex of Escherichia coli, by an extensive cysteine scanning mutagenesis of residues likely involved in ligand transport. Many such mutations in MdtC strongly decreased the level of cloxacillin transport, whereas mutations of corresponding residues in MdtB did not affect transport. Furthermore, many such residues in MdtC were covalently modified by fluorescein maleimide, which acted as a substrate and presumably produced labeling of the residues in the substrate path. In contrast, few residues in MdtB were labeled. Together with the previous data showing that the inactivation of proton translocation channel in MdtC has an only modest effect on transport yet in MdtB totally inactivated the activity, these results suggest that the two subunits, MdtB and MdtC, play very different roles, MdtC likely involved in substrate binding and transport and MdtB presumably inducing the conformational change needed for transport through proton translocation. Three-dimensional models of MdtB and MdtC, based on sequence homology with the AcrB transporter, also support this interpretation.     

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V₁ domain that hydrolyzes ATP and an integral V₀ domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V₀) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V₁V₀ complex and the free V₀ domain to identify residues protected from modification by the presence of V₁. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V₁ domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.     

Reversible inhibition of the mitochondrial ubiquinol-cytochrome c oxidoreductase complex (complex III) by ethoxyformic anhydride

The mitochondrial ubiquinol-cytochrome c oxidoreductase (complex III) is inhibited by ethoxyformic anhydride (EFA). The inhibition is readily reversed by hydroxylamine, suggesting the involvement of essential histidyl or possibly tyrosyl residues. The spectrum of ethoxyformylated complex III in the UV region showed a peak at 238 nm, indicative of N-(ethoxyformyl)histidine. Addition of hydroxylamine caused a large decrease of the 238-nm peak, which amounted to 16 mol of (ethoxyformyl)histidine/mol of cytochrome c1. Hydroxylamine addition to ethoxyformylated complex III also caused a small change at about 280 nm, which could be due to reversal of 1.6 O-ethoxyformylated tyrosyl residues/mol of cytochrome c1. Among many inhibitors of the cytochrome bc1 region of the respiratory chain, EFA is the only reagent known to cause reversible inhibition by covalent modification of amino acid residues. The inhibition site of EFA was determined to be between cytochromes b-562 and c1. However, unlike antimycin, which also inhibits in the same region, EFA did not promote the reduction of cytochrome b-566 in particles treated with substrates. In addition, it was found that EFA inhibits proton translocation in the cytochrome bc1 region and is a more effective electron transport inhibitor when added to reduced particles as compared to oxidized particles. These results together with the strong possibility that the EFA target is a histidyl or possibly a tyrosyl residue have been discussed in relation to the mechanism of proton translocation by complex III.     

Effects of mutations of Lys41 and Asp102 of bacteriorhodopsin

Bacteriorhodopsin (BR) is a retinal protein that functions as a light-driven proton pump. In this study, six novel mutants including K41E and D102K, were obtained to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake, because we found that the order was reversed in another retinal protein archaerhodopsin 4 (AR4), which had different 41th and 102th residues. Our results rule out that possibility and confirm that the pK(a) of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pK(a) of Asp96. Compared to the wild-type BR (BR-WT), the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor.     

The role of tyrosine residues in the RNA N-glycosidase activity of cinnamomin A-chain

Cinnamomin is a type II ribosome-inactivating protein (RIP) and its A-chain (CTA) is a RNA N-glycosidase. It is observed that modification of tyrosine residues by N-acetylimidazole (N-AI) causes almost complete loss of CTA activity. Adenine partially protects tyrosine residues from modification by N-AI. It is proposed that tyrosine residues are involved in the active site of CTA and they are crucial in recognition and binding of ribosomal RNA. Tryptophan residues of CTA are also studied by NBS modification.     

Sequence-specific 1H-NMR assignments and secondary structure of the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex

The lipoyl domain (residues 1-85) of the lipoate-acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been subjected to detailed structural analysis by means of two-dimensional (2D) 1H-NMR spectroscopy at 400 MHz. Sequence-specific proton resonance assignments were made, but at this field strength not all of the side-chain protons could be assigned, especially from complex spin systems like those of leucine, proline and lysine residues. Measurement of short-range interproton distances identified two extensive regions of beta-sheet, each containing four anti-parallel peptide strands. The lipoyl-lysine residue (Lys42) is located in a tight turn at a corner of one sheet, the N-terminal and C-terminal residues of the domain are close together in two adjacent beta-strands in the other. The lipoylated and unlipoylated forms of the domain have almost identical spectra, indicating that there is little, if any, conformational change in the protein as a result of the post-translational modification.     

Effects of Mutations of Lys41 and Asp102 of Bacteriorhodopsin

Bacteriorhodopsin (BR) is a retinal protein that functions as a light-driven proton pump. In this study, six novel mutants including K41E and D102K, were obtained to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake, because we found that the order was reversed in another retinal protein archaerhodopsin 4 (AR4), which had different 41th and 102th residues. Our results rule out that possibility and confirm that the pK _a of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pK _a of Asp96. Compared to the wild-type BR (BR-WT), the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor.     

Ras (CXXX) and Rab (CC/CXC) prenylation signal sequences are unique and functionally distinct.

Rab proteins typically lack the consensus carboxyl-terminal CXXX motif that signals isoprenoid modification of Ras and other isoprenylated proteins and, instead, terminate in either CC or CXC sequences (C = cysteine, X = any amino acid). To compare the functional relationship between the Ras CXXX and the Rab CC/CXC motifs, we have generated chimeric Ras proteins terminating in Rab carboxyl-terminal CC or CXC sequences. These mutant Ras proteins were not isoprenylated in vitro or in vivo, demonstrating that the CC and CXC sequences alone are not sufficient to replace a CXXX sequence to signal Ras isoprenoid modification. Surprisingly, chimeric Ras/Rab proteins terminating in significant lengths of carboxyl-terminal sequences from Rab1b (7-139 residues), Rab2 (5-151 residues), or Rab3a (12 residues) were also not isoprenylated. These results demonstrate that the sequence requirements for isoprenoid modification of Rab proteins are more complex than the simple tetrapeptide CXXX sequence for isoprenoid modification of Ras proteins and suggest that the Rab geranylgeranyl transferase(s) requires recognition of protein conformation to signal the addition of geranylgeranyl groups. Finally, competition studies demonstrate that a common geranylgeranyl transferase activity is responsible for the modification of Rab proteins terminating in CC or CXC motifs.