Lawrence J. Prochaska

On the role of subunit III in proton translocation in cytochromec oxidase

Mammalian mitochondrial cytochromec oxidase catalyzes the transfer of electrons from ferrocytochromec to molecular oxygen in the respiratory chain, while conserving the energy released during its electron transfer reactions by the vectorial movement of protons across the inner membrane of the mitochondrion. The protein domain that translocates the protons across the membrane is currently unknown. Recent research efforts have investigated the role of one of the transmembrane subunits of the enzyme (III,Mr 29,884) in the vectorial proton translocation reaction. The data that favor subunit III as integral in vectorial proton translocation as well as the data that support a more peripheral role for subunit III in proton translocation are reviewed. Possible experimental approaches to clarify this issue are presented and a general model discussed.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase and subunit III-deficient enzyme: analysis of respiratory control and proton translocating activities.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) or subunit III (Mr 29884)-deficient enzyme (COV-III) were characterized for electron transfer and proton translocating activities in order to investigate the relationship between the respiratory control ratio (RCR) and the apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) in these preparations. We did not observe a quantitative correlation between the RCR value and the H+/e- ratio in the preparations. Significant deviation between these two parameters was observed in COV-III and also in COV. However, a new parameter, RCRval, did show a linear relationship with the H+/e- ratio of each preparation. Subunit III (SIII)-deficient cytochrome c oxidase isolated by either native gel electrophoresis or chymotrypsin treatment and incorporated into COV-III exhibited H+/e- ratios of 0.34 +/- 0.10, compared to 0.63 +/- 0.09 for COV, emphasizing that the 50% decrease of proton translocating activity is independent of the method of removal of SIII from the enzyme. COV and COV-III also showed similar rates of alkalinization of the extravesicular media after the initial proton translocation reaction (0.07-0.09 neq OH-/s), suggesting that these two preparations had similar endogenous proton permeabilities. In contrast, cytochrome c oxidase (COX) treated with Triton X-100 (3 mg/mg COX) and incorporated into phospholipid vesicles [COV (+TX)] exhibited slower rates of alkalinization (0.04 neq OH-/s), while having a H+/e- ratio similar to that of COV (0.66 +/- 0.10). The passive proton permeabilities of these preparations were tested by valinomycin-induced K+/H+ exchange activity. COV (+TX) and COV-III exhibited similar pseudo-first-order rate constants (10 peq OH-/s), while COV had a 20-fold higher rate constant. These results taken together suggest that the different preparations of COX-containing phospholipid vesicles have different biophysical properties. In addition, the decrease in proton-pumping activity observed in COV-III is due to removal of SIII from COX, suggesting that SIII may act either as a passive proton-conducting channel or as a regulator of COX conformation and/or functional activities.

Cardiac nonmyofibrillar proteins in copper-deficient rats

Cardiac nonmyofibrillar proteins from copper-deficient rats appear to have diminished quantity of selected peptides. Identification of some of these peptides was the objective of the present study. Male weanling Long-Evans rats were fed either copper-adequate (n=6) or copper-deficient (n=6) diets for 5 wk. At the end of 5 wk, the rat hearts were removed, quick frozen in liquid nitrogen, and non-myofibrillar proteins separated using sodium-dodecyl-sulfate poly-acrylamide gel electrophoresis (SDS-PAGE). A peptide in the 16-kDa mol-wt region was diminished in copper-deficient rats. Blotting of gels to an Immobilon-P membrane and subsequent sequencing of the amino acids identified the peptide as the δ subunit of mitochondrial ATP synthase. Blotting of gels to nitrocellulose followed by Western blot assay for cytochrome C oxidase using antibodies against the enzyme complex revealed decreased protein content in the copper-deficient rat for this enzyme, primarily the nuclear encoded subunits.

Cardiac levels of fibronectin, laminin, isomyosins, and cytochrome c oxidase of weanling rats are more vulnerable to copper deficiency than those of postweanling rats

The relative quantities of cardiac laminin, fibronectin, cytochrome c oxidase (CCO), and isomyosin types were studied by gel electrophoresis in male rats fed copper-deficient diets beginning either from the time of weaning for 5 weeks or from 5 weeks postweaning for 6 weeks with one group of copper-repleted rats. Increased levels of fibronectin and V(3) isomyosin but decreased levels of CCO subunit IV and laminin were found in weanling copper-depleted rats. In contrast, postweanling copper-depleted rats exhibited only increased levels of fibronectin and decreased levels of cardiac CCO subunit IV. Repletion of copper-deficient rats for 6 weeks was not sufficient to restore CCO subunit IV to the same level as controls. These results confirm that biochemical lesions in the basal laminae are a result of copper restriction. The decreased nuclear encoded subunits of CCO may help explain some of the mitochondrial pathology observed in dietary copper restriction. Increased V(3) isomyosin levels with low ATPase activity may help to conserve to a limited extent the ATP levels in copper-deficient cardiac tissue. These protein changes are consistent with the known morphological alterations of hearts from copper-restricted rats.

Site-specific antibodies against hydrophilic domains of subunit III of bovine heart cytochrome c oxidase affect enzyme function

Antibodies were raised against conserved amino acid sequences in four extramembranous portions of subunit III (sIII) from beef cytochrome c oxidase (COX) and the role of these domains in the functional activities of the enzyme was investigated. The binding of one antipeptide antibody corresponding to an externally exposed (facing the intermembrane space) domain of COX sIII (amino acids 180-189 in the primary sequence) exhibited a 30-50% stimulation of electron transfer activity in both detergent-dispersed COX and COX incorporated into phospholipid vesicles (COV). Antibody binding to two different matrix-faced domains (amino acids 57-66 and 148-159 in the sequence) resulted in small stimulations (10-25%) of COX electron transfer activity. The remaining antipeptide antibody (against amino acids 119-128) had no effect on electron transfer activity of COX in detergent solution, but exhibited a slight inhibition of activity (15%) in COV. The mechanism of antibody-induced stimulation of COX electron transfer activity was determined to be an increase in the maximum velocity of the enzyme and not due to a change in the apparent K(m) of cytochrome c interaction with COX as determined by steady state kinetic assays. Antibody binding to COX in COV increased the respiratory control ratio (an indicator of endogenous proton permeability) of COV, but had no effect on the vectorial proton pumping activity of COV. These results suggest that these conserved, hydrophilic domains of COX sIII are conformationally linked to the electron transfer function of the enzyme in subunits I and II and that sIII may serve as a regulatory subunit for COX electron transfer and proton pumping activities.

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase☆

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.

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.

Role of the N-terminus of subunit III in proton uptake in cytochrome c oxidase of Rhodobacter sphaeroides.

The catalytic core of cytochrome c oxidase consists of three subunits that are conserved across species. The N-terminus of subunit III contains three histidine residues (3, 7, and 10) that are surface-exposed, have physiologically relevant pKa values, and are in close proximity of the mouth of the D-channel in subunit I. A triple-histidine mutation (to glutamine) was created in Rhodobacter sphaeroides. The mutant enzyme retains 60% of wild-type activity. Absorbance during steady-state turnover indicates that electrons accumulate at heme a in the mutant, accompanied by accumulation of the oxoferryl intermediate. When reconstituted into liposomes, the mutant enzyme pumps protons with an efficiency that is half that of the wild type. Finally, the mutant exhibits a lower cytochrome c peroxidation rate. Our results indicate that the mutation lowers activity indirectly by slowing the uptake of protons through the D-channel and that the three histidine residues stabilize the interactions between subunit I and subunit III.

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3′‐end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa 3‐type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.

Biochemical and biophysical properties of purified phospholipid vesicles containing bovine heart cytochrome c oxidase

Liposomes containing bovine heart cytochrome c oxidase (COV) prepared by the cholate dialysis technique were purified from those devoid of the enzyme using discontinuous sucrose density ultra centrifugation to eliminate interference in proton-pumping assays. This technique was also used to purify liposomes containing cytochrome c oxidase depleted in subunit III (COV-III), a COX enzyme preparation with altered subunit structure, to assess if the technique could be applied to COX enzymes in which structural and functional changes have occurred. Upon discontinuous sucrose density ultra gradient ultracentrifugation, either COV or COV-III were separated into two bands. Liposomes devoid of enzyme sedimented into the 12% sucrose layer, whereas enzyme-containing liposomes (pCOV or pCOV-III) were found in the 13% sucrose layer. The yield of both pCOV or pCOV-III was greater than 60% (based on heme aa(3) content), suggesting a similar distribution of cytochrome c oxidase (COX) and subunit III-depleted enzyme (COX-III) in the purified liposomes. The number of COX or COX-III molecules per phospholipid vesicle in purified fractions was estimated to be two. Removal of subunit III (M(r)=29,918) from COX resulted in a 30% decrease in electron transfer activity (either in COV-III or pCOV-III) when compared with COV and pCOV, respectively. Both pCOV and pCOV-III exhibited low endogenous proton permeability, as assessed by possessing high respiratory control ratios (14 and greater) and by having similar valinomycin concentration dependencies for stimulation of electron transfer activity in the presence of saturating amounts of CCCP. COV-III and pCOV-III exhibited a 39-44% decrease in proton-pumping activity when compared with COV and pCOV. These results showed that the separation of COX containing liposomes from those lacking enzyme by sucrose density gradient centrifugation can be used to characterize the biophysical properties of these liposomes.