Spectroscopic and functional properties of subunit III-depleted cytochrome oxidase

Beef heart cytochrome c oxidase has been depleted of subunit III by treatment with chymotrypsin. The removal of subunit III has been evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel fluorography of preparations of the oxidase labeled with [14C]dicyclohexylcarbodiimide prior to proteolysis. Removal of subunit III resulted in a perturbation of the visible spectrum of reduced cytochrome oxidase. Subunit III-depleted oxidase is spectroscopically very similar to the oxidase from Paracoccus denitrificans. When reconstituted into liposomes, the depleted enzyme still pumped protons in response to a pulse of reduced cytochrome c. The H+/e- stoichiometry averaged 0.5. Redox-linked proton translocation could be observed only when respiratory control ratios were higher than 3 and the reductant pulse was of a magnitude that allowed for no more than 5 turnovers of the oxidase.     

Dicyclohexylcarbodiimide binds specifically and covalently to cytochrome c oxidase while inhibiting its H+-translocating activity

We have investigated the covalent binding of dicyclohexylcarbodiimide (DCCD) to cytochrome c oxidase in relation to its inhibition of ferrocytochrome c-induced H+ translocation by the enzyme reconstituted in lipid vesicles. DCCD bound to the reconstituted oxidase in a time- and concentration-dependent manner which appeared to correlate with its inhibition of H+ translocation. In both reconstituted vesicles and intact beef heart mitochondria, the DCCD-binding site was located in subunit III of the oxidase. The apolar nature of DCCD and relatively minor effects of the hydrophilic carbodiimide, 1-ethyl-(3-dimethylaminopropyl)-carbodiimide, on H+ translocation by the oxidase indicate that the site of action of DCCD is hydrophobic. DCCD also bound to isolated cytochrome c oxidase, though in this case subunits III and IV were labeled. The maximal overall stoichiometries of DCCD molecules bound per cytochrome c oxidase molecule were 1 and 1.6 for the reconstituted and isolated enzymes, respectively. These findings point to subunit III of cytochrome c oxidase having an important role in H+ translocation by the enzyme and indicate that DCCD may prove a useful tool in elucidating the mechanism of H+ pumping.     

Derivatization of the catalytic subunits of the chloroplast ATPase by 2-azido-ATP and dicyclohexylcarbodiimide. Evidence for catalytically induced interchange of the subunits

Modifications of the catalytic beta subunits of the chloroplast ATPase (CF1-ATPase) are reported which support the proposal that all three subunits participate sequentially during catalysis. The beta subunits of the CF1-ATPase are sufficiently homogeneous to allow detection of their derivatization with dicyclohexylcarbodiimide (DCCD) or the substrate analog 2-azido-ATP by two-dimensional isoelectric focusing. Whether the DCCD reacts with the same beta subunit that tightly binds ATP has not been known. Our results show that when CF1-ATPase is covalently labeled with 2-azido-ATP followed by reaction with DCCD, different beta subunits are labeled. The DCCD labeling does not stop catalytic cooperativity of the CF1-ATPase and allows slow enzyme turnover. When the DCCD-modified enzyme catalyzes 2-azido-ATP cleavage and the enzyme with tightly bound nucleotide is photolyzed, both DCCD-modified and unmodified subunits are randomly labeled by the azido nucleotide. This result is as expected if during the catalytic cycle one beta subunit with unique properties is replaced by another subunit that gains these properties. The participation of all three subunits in the catalytic cycle is suggested by the apparent retention of catalytic cooperativity by the two remaining subunits after one subunit has already catalyzed 2-azido-ATP cleavage and been labeled.     

Characterization of electron-transfer and proton-translocation activities in bovine heart mitochondrial cytochrome c oxidase deficient in subunit III

The electron-transfer and proton-translocation activities of cytochrome c oxidase deficient in subunit III (Mr 29 884) prepared by native gel electrophoresis [Ludwig, B., Downer, N. W., & Capaldi, R. A. (1979) Biochemistry 18, 1401-1407] have been investigated. This preparation has been depleted of 82-87% of its subunit III content as quantitated by Coomassie Brilliant Blue staining intensity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and [14C]dicyclohexylcarbodiimide labeling. The maximum rate of electron transfer of the subunit III deficient enzyme at pH 6.5 is 383 s-1, 78% of control enzyme. Neither the high-affinity site (Km = 10(-8) M) nor the low-affinity site (Km = 10(-6) M) of the cytochrome c kinetic interaction with cytochrome c oxidase is affected by the removal of subunit III. Subunit III deficient cytochrome c oxidase retains the ability to bind cytochrome c in both the high- and low-affinity sites as determined in direct thermodynamic binding experiments. Liposomes containing this preparation exhibit a respiratory control ratio [Hinkle, P. C., Kim, J. J., & Racker, E. (1972) J. Biol. Chem. 247, 1338-1341] of 3.9, while liposomes containing control enzyme exhibit a ratio of 4.3, suggesting that they have a similar proton permeability. Vectorial proton translocation initiated by the addition of ferrocytochrome c in liposomes containing subunit III deficient enzyme is decreased by 64% compared to those containing control enzyme. When the proton-translocated to electron-transferred ratio is measured in these phospholipid vesicles at constant enzyme turnover, removal of subunit III from the enzyme decreases the ratio from 0.52 to 0.21, a 60% decrease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the F0 portion of the H+-translocating adenosinetriphosphatase complex of Escherichia coli by the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and effect on the proton channeling function

1-Ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), a water-soluble carbodiimide, inhibited ECF1-F0 ATPase activity and proton translocation through F0 when reacted with Escherichia coli membrane vesicles. The site of modification was found to be in subunit c of the F0 portion of the enzyme but did not involve Asp-61, the site labeled by the hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD). EDC was not covalently incorporated into subunit c in contrast to DCCD. Instead, EDC promoted a cross-link between the C-terminal carboxyl group (Ala-79) and a near-neighbor phosphatidylethanolamine as evidenced by fragmentation of subunit c with cyanogen bromide followed by high-pressure liquid chromatography and thin-layer chromatography.     

Dicyclohexylcarbodiimide cross-links two conserved residues, Asp-184 and Lys-72, at the active site of the catalytic subunit of cAMP-dependent protein kinase

In the absence of MgATP, the catalytic subunit of cAMP-dependent protein kinase is irreversibly inhibited by the hydrophobic carbodiimide dicyclohexylcarbodiimide, and this inhibition is most likely due to the formation of a cross-link between a carboxyl group and a lysine residue in the active site (Toner-Webb & Taylor, 1987). In order to identify these cross-linked residues, the catalytic subunit was modified by dicyclohexylcarbodiimide and then treated with acetic anhydride and digested with trypsin. The resulting peptides were resolved by high-performance liquid chromatography. One major absorbing tryptic peptide and one smaller peptide consistently and reproducibly showed a decrease in absorbance after the catalytic subunit had been treated with DCCD. These peptides correspond to residues 166-190 and 57-93, respectively. A unique peptide was isolated from the modified catalytic subunit, and the sequence of this peptide established that the cross-linking occurred between Asp-184 and Lys-72. The cross-linking of these two residues, which were both identified previously as essential residues, confirms the likelihood that each plays a role in the functioning of this enzyme. The fact that Asp-184 and Lys-72 appear to be invariant in all protein kinases further supports the hypothesis that these two residues, located close to one another at the active site of the enzyme, play essential roles in catalysis.     

Labeling of individual amino acid residues in the membrane-embedded F0 part of the F1 F0 ATP synthase from Neurospora crassa. Influence of oligomycin and dicyclohexylcarbodiimide

Three F0 subunits and the F1 subunit beta of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confined to five residues at the NH2-terminus and five residues at the C-terminus of the protein. Labeling occurred at similar positions compared with the homologous protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms. The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent of labeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodiimide-binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu-65 is located at the protein-lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F0 subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F0 subunits.     

Disposition of polar and nonpolar residues on outer surfaces of transmembrane helical segments of proteins involved in proton translocation

"Helical wheel" projections of transmembrane helical segments of membrane proteins involved in proton translocation were constructed. The particular proteins studied were the uncF protein subunit of the Escherichia coli proton-ATPase, the uncE protein subunit of the E. coli proton-ATPase, and cytochrome oxidase subunit III. Clear demarcation of polar and nonpolar regions on surfaces of transmembrane helical segments was seen in the uncF protein and in uncE protein helical segment two, but not in uncE protein helical segment one. The transmembrane segment of cytochrome oxidase subunit III which includes the dicyclohexylcarbodiimide (DCCD)-reactive residue was very similar to E. coli uncE protein helical segment two. The DCCD-reactive residue in both was clearly located on a nonpolar surface.     

Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.     

Membrane-protein structural mapping of chloroplast coupling factor in asolectin vesicles

The spatial relationship of specific sites on chloroplast coupling factor, reconstituted in asolectin vesicles, to the bilayer surface has been studied with fluorescence methods. Fluorescence resonance energy transfer measurements have been used to map the distances of closest approach of the N,N'-dicyclohexylcarbodiimide-binding site and the disulfide on the gamma-polypeptide to the bilayer center. The dicyclohexylcarbodiimide site was labeled with N-cyclohexyl-N'-pyrenylcarbodiimide and the gamma-disulfide site with a coumarinyl derivative. The bilayer center was labeled with 25-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-N-methylamino]-27-norc holesterol. The distances obtained, 15 and 43 A, respectively, were combined with previous measurements of the distance of closest approach between these sites and the membrane surface to estimate the perpendicular distances of the sites from the membrane surface. The depth of the dicyclohexylcarbodiimide site was also determined by studying the quenching of fluorescence by 5-, 7-, 12-, and 16-doxylstearic acids. The model developed suggests that the dicyclohexylcarbodiimide site is 6-10 A below the membrane surface and the gamma-disulfide is 16 A above the membrane surface. The distances measured are subject to a considerable uncertainty, but the proposed model provides a useful starting point for further structural studies.     

Fluorescence labeling of proteins related to ADH-induced change in frog bladder luminal membrane

Sulfhydryl (SH) reactive reagents, such as eosin derivatives, have been found to be useful in labeling water pathways in red cells. In the present study we used an impermeable SH-reagent, a fluorescent maleimide analogue EMA (eosin-5'-maleimide), in order to identify proteins involved in water permeability response to antidiuretic hormone (ADH). We observed that: 1) EMA (1 mM) mucosal pretreatment did not modify either the basal water flux or the subsequent ADH-induced hydrosmotic response; 2) EMA added to the mucosal bath at the maximum response to ADH, significantly decreased net water flux by about 40%; similar results were obtained when 10(-5) M forskolin was used as a hydrosmotic agent. These results suggest that the inhibitory effect of EMA occurs at a post cAMP step, possibly at the level of the sulfhydryl groups of the water channels themselves. Fluorescence distribution in SDS-PAGE of Triton X-100 extracted proteins from bladder labeled with EMA in both control conditions and under ADH stimulation allowed us to identify apical membrane proteins, labeled during ADH stimulation and not labeled in water impermeable controls. Of particular importance are four proteins of 52, 32-35, 26, 17, kDa. These polypeptides are probably involved in ADH-stimulated water transport and may be components of the water channels.     

Coupled rotation within single F0F1 enzyme complexes during ATP synthesis or hydrolysis

F0F1 ATP synthases are the smallest rotary motors in nature and work as ATP factories in bacteria, plants and animals. Here we report on the first observation of intersubunit rotation in fully coupled single F0F1 molecules during ATP synthesis or hydrolysis. We investigate the Na+-translocating ATP synthase of Propionigenium modestum specifically labeled by a single fluorophore at one c subunit using polarization-resolved confocal microscopy. Rotation during ATP synthesis was observed with the immobilized enzyme reconstituted into proteoliposomes after applying a diffusion potential, but not with a Na+ concentration gradient alone. During ATP hydrolysis, stepwise rotation of the labeled c subunit was found in the presence of 2 mM NaCl, but not without the addition of Na+ ions. Moreover, upon the incubation with the F0-specific inhibitor dicyclohexylcarbodiimide the rotation was severely inhibited.     

Dicyclohexylcarbodiimide binds to cytochrome b and subunit VIII in soluble complex III from beef heart mitochondria

Incubation of soluble complex III isolated from either yeast or beef heart mitochondria with 25-100 nmol of [14C]dicyclohexylcarbodiimide (DCCD)/nmol of cytochrome b followed by centrifugation through 10% sucrose or precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunits of either complex. The [14C]DCCD was bound to cytochrome b and phospholipids in the yeast complex and with similar kinetics to both cytochrome b and subunit VIII (Mr = 4000-8000) plus phospholipids of the beef complex. Subunit VIII of the beef complex was partially extracted with chloroform:methanol; however, no subunit of this mobility was present in the yeast complex. Incubation of the beef complex in phosphate buffer for short times resulted in a doubling of the [14C]DCCD bound to cytochrome b relative to that to subunit VIII. Preincubation of both complexes with venturicidin prior to treatment with DCCD resulted in a 50% decrease in the binding of [14C]DCCD to cytochrome b. Reisolation of the beef complex III by precipitation with (NH4)2SO4 after incubation with [14C]DCCD resulted in the formation of a new band with an apparent molecular weight of 39,000 even in the zero time control. The [14C]DCCD was bound to subunit VIII and the core proteins but not to cytochrome b at all times, suggesting that precipitation with (NH)2SO4 in the presence of DCCD causes cross-linking of the subunits of complex III.     

Fluorescence quenching of reconstituted NCD-4-labeled cytochrome c oxidase complex by DOXYL-stearic acids.

It has been known for some time that dicyclohexylcarbodiimide (DCCD) inhibits the proton translocation function of the cytochrome c oxidase complex (CcO) and that there is one major site in subunit III which is modified upon reaction with DCCD (Glu-90 for the bovine enzyme). We have examined the reaction of bovine CcO with N-cyclohexyl-N'-(4-dimethylamino-alpha-napthyl)carbodiimide (NCD-4), a fluorescent analog of DCCD. NCD-4 labeling of CcO is strongly inhibited by DCCD implicating Glu-90 of subunit III as the site of chemical modification by NCD-4. The fluorescence of reconstituted NCD-4-labeled bovine CcO is strongly quenched by hydrophobic nitroxides, whereas hydrophilic nitroxides and iodide ions have a reduced quenching ability. It is concluded that the Glu-90 of subunit III resides near the protein-lipid interface of the membrane spanning region of the enzyme. Different quenching abilities of 5-, 7-, 10-, 12-, and 16-4,4-dimethyl-3-oxazolinyloxy-stearic acids suggest that the NCD-4 label is located in the membrane bilayer in the region near the middle of the hydrocarbon tail of stearic acid. In light of these results, it is unlikely that Glu-90 is part of a proton channel that is associated with the proton pumping machinery of the enzyme but the outcome of this study does not eliminate an allosteric regulatory role for this residue.     

Insights into the rotary catalytic mechanism of F0F1 ATP synthase from the cross-linking of subunits b and c in the Escherichia coli enzyme

The transmembrane sector of the F(0)F(1) rotary ATP synthase is proposed to organize with an oligomeric ring of c subunits, which function as a rotor, interacting with two b subunits at the periphery of the ring, the b subunits functioning as a stator. In this study, cysteines were introduced into the C-terminal region of subunit c and the N-terminal region of subunit b. Cys of N2C subunit b was cross-linked with Cys at positions 74, 75, and 78 of subunit c. In each case, a maximum of 50% of the b subunit could be cross-linked to subunit c, which suggests that either only one of the two b subunits lie adjacent to the c-ring or that both b subunits interact with a single subunit c. The results support a topological arrangement of these subunits, in which the respective N- and C-terminal ends of subunits b and c extend to the periplasmic surface of the membrane and cAsp-61 lies at the center of the membrane. The cross-linking of Cys between bN2C and cV78C was shown to inhibit ATP-driven proton pumping, as would be predicted from a rotary model for ATP synthase function, but unexpectedly, cross-linking did not lead to inhibition of ATPase activity. ATP hydrolysis and proton pumping are therefore uncoupled in the cross-linked enzyme. The c subunit lying adjacent to subunit b was shown to be mobile and to exchange with c subunits that initially occupied non-neighboring positions. The movement or exchange of subunits at the position adjacent to subunit b was blocked by dicyclohexylcarbodiimide. These experiments provide a biochemical verification that the oligomeric c-ring can move with respect to the b-stator and provide further support for a rotary catalytic mechanism in the ATP synthase.     

Structure-function investigations of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B: studies of reactive amino acid residues using group-specific reagents

The purified, lipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B was treated with a variety of reagents which specifically modify various amino acid residues on the enzyme. In all cases reaction of this enzyme with any of the reagents tested results in at least a partial inactivation of its activity. The modification of one reactive lysine by dinitrofluorobenzene, of one reactive arginine by phenylglyoxal, or of two tyrosine residues by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or fluorosulfonylbenzoyl adenosine results in a complete inactivation of the enzyme. Partial inactivation of enzymatic activity with N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, dicyclohexylcarbodiimide, and Woodward's reagent K suggests an indirect involvement of sulfhydryl and carboxylic acid groups in the maintenance of enzymatic activity, although inhibition by these reagents may also be the result of nonspecific effects such as subunit crosslinking. These studies also show that all of the subunits of the ATPase can be labeled by aqueous-phase reagents directed at amino groups and phenolic groups, and provide evidence for a specific affinity labeling of the alpha subunit of the enzyme by a nucleotide analog directed at phenolic and/or sulfhydryl groups.     

Biochemical properties of the PsbS subunit of photosystem II either purified from chloroplast or recombinant

The biochemical properties of PsbS protein, a nuclear-encoded Photosystem II subunit involved in the high energy quenching of chlorophyll fluorescence, have been studied using preparations purified from chloroplasts or obtained by overexpression in bacteria. Despite the homology with chlorophyll a/b/xanthophyll-binding proteins of the Lhc family, native PsbS protein does not show any detectable ability to bind chlorophylls or carotenoids in conditions in which Lhc proteins maintain full pigment binding. The recombinant protein, when refolded in vitro in the presence of purified pigments, neither binds chlorophylls nor xanthophylls, differently from the homologous proteins LHCII, CP26, and CP29 that refold into stable pigment-binding complexes. Thus, it is concluded that if PsbS is a pigment-binding protein in vivo, the binding mechanism must be different from that present in other Lhc proteins. Primary sequence analysis provides evidence for homology of PsbS helices I and III with the central 2-fold symmetric core of chlorophyll a/b-binding proteins. Moreover, a structural homology owed to the presence of acidic residues in each of the two lumen-exposed loops is found with the dicyclohexylcarbodiimide/Ca(2+)-binding domain of CP29. Consistently, both native and recombinant PsbS proteins showed [(14)C]dicyclohexylcarbodiimide binding, thus supporting a functional basis for its homology with CP29 on the lumen-exposed loops. This domain is suggested to be involved in sensing low luminal pH.     

Evidence for a Conformational Change in Subunit III of Bovine Heart Mitochondrial Cytochrome c Oxidase1

The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl—biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.     

Topological studies of monomeric and dimeric cytochrome c oxidase and identification of the copper A site using a fluorescence probe

Beef heart cytochrome c oxidase was labeled at a single sulfhydryl group by treatment with 5 mM N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS) at pH 8.0 for 4 h. Sodium dodecyl sulfate gel electrophoresis revealed that the enzyme was exclusively labeled at subunit III, presumably at Cys-115. The high affinity phase of the electron transfer reaction with horse cytochrome c was not affected by acetylamidoethyl-1-aminonaphthalene-5-sulfonate (AEDANS) labeling. Addition of horse cytochrome c to dimeric AEDANS-cytochrome c oxidase resulted in a 55% decrease in the AEDANS fluorescence due to the formation of a 1:1 complex between the two proteins. Forster energy transfer calculations indicated that the distance from the AEDANS label on subunit III to the heme group of cytochrome c was in the range 26-40 A. In contrast to the results with the dimeric enzyme, the fluorescence of monomeric AEDANS-cytochrome c oxidase was not quenched at all by binding horse heart cytochrome c, indicating that the AEDANS label on subunit III was at least 54 A from the heme group of cytochrome c. These results support a model in which the lysines surrounding the heme crevice of cytochrome c interact with carboxylates on subunit II of one monomer of the cytochrome c oxidase dimer and the back of the molecule is close to subunit III on the other monomer. In order to identify the cysteine residues that ligand copper A, a new procedure was developed to specifically remove copper A from cytochrome c oxidase by incubation with 2-mercaptoethanol followed by gel chromatography. Treatment of the copper A-depleted cytochrome c oxidase preparation with 1,5-I-AEDANS resulted in labeling sulfhydryl groups on subunit II as well as on subunit III. No additional subunits were labeled. This result indicates that the copper A binding site is located at cysteines 196 and/or 200 of subunit II and that removal of copper A exposes these residues for labeling by 1,5-I-AEDANS. Alternative copper A depletion methods involving incubation with bathocuproine sulfonate (Weintraub, S.T., and Wharton, D.C. (1981) J. Biol. Chem. 256, 1669-1676) or p-(hydroxymercuri)benzoate (Li, P.M., Gelles, J., Chan, S.I., Sullivan, R.J., and Scott, R.A. (1987) Biochemistry 26, 2091-2095) were also investigated. Treatment of these preparations with 1,5-I-AEDANS resulted in labeling cysteine residues on subunits II and III. However, additional sulfhydryl residues on other subunits were also labeled, preventing a definitive assignment of the location of copper A using these depletion procedures.     

Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis.

In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site.