The metalloclusters of carbon monoxide dehydrogenase/acetyl-CoA synthase: a story in pictures

Eight Ni proteins are known and three of these, CO dehydrogenase (CODH), acetyl-CoA synthase (ACS), and hydrogenase, are Ni-Fe-S proteins. In the last three years, the long-awaited structures of CODH and ACS have been solved. The bioinorganic community was shocked, as the structures of the active sites of CODH and ACS, the C- and A-cluster, respectively, which each had been predicted to consist of a [Fe₄S₄] cluster bridged to a single Ni, revealed unexpected compositions and arrangements. Crystal structures of ACS revealed major differences in protein conformation and in A-cluster composition; for example, a [Fe₄S₄] cluster bridged to a binuclear center in which one of the metal binding sites was occupied by Ni, Cu, or Zn. Recent studies have revealed Ni-Ni to be the active state, unveiled the source of the heterogeneity that had plagued studies of CODH/ACS for decades, and produced a metal-replacement strategy to generate highly active and nearly homogeneous enzyme.Abbreviations CFeSP corrinoid iron-sulfur protein - CH₃H₄folate methyltetrahydrofolate - CODH/ACS carbon monoxide dehydrogenase/acetyl-CoA synthases - ENDOR electron nuclear double resonance - MeTr methyltransferase     

Contrasting Effects of Cd<Superscript>2+</Superscript> and Co<Superscript>2+</Superscript> on the Blocking/Unblocking of Human Ca<Subscript>v</Subscript>3 Channels

Inorganic ions have been used widely to investigate biophysical properties of high voltage-activated calcium channels (HVA: Ca_v1 and Ca_v2 families). In contrast, such information regarding low voltage-activated calcium channels (LVA: Ca_v3 family) is less documented. We have studied the blocking effect of Cd²⁺, Co²⁺ and Ni²⁺ on T-currents expressed by human Ca_v3 channels: Ca_v3.1, Ca_v3.2, and Ca_v3.3. With the use of the whole-cell configuration of the patch-clamp technique, we have recorded Ca²⁺ (2 mM) currents from HEK−293 cells stably expressing recombinant T-type channels. Cd²⁺ and Co²⁺ block was 2- to 3-fold more potent for Ca_v3.2 channels (EC₅₀ = 65 and 122 μM, respectively) than for the other two LVA channel family members. Current-voltage relationships indicate that Co²⁺ and Ni²⁺ shift the voltage dependence of Ca_v3.1 and Ca_v3.3 channels activation to more positive potentials. Interestingly, block of those two Ca_v3 channels by Co²⁺ and Ni²⁺ was drastically increased at extreme negative voltages; in contrast, block due to Cd²⁺ was significantly decreased. This unblocking effect was slightly voltage-dependent. Tail-current analysis reveals a differential effect of Cd²⁺ on Ca_v3.3 channels, which can not close while the pore is occupied with this metal cation. The results suggest that metal cations affect differentially T-type channel activity by a mechanism involving the ionic radii of inorganic ions and structural characteristics of the channels pore.     

Temporal variability in <Superscript>13</Superscript>C of respired CO<Subscript>2</Subscript> in a pine and a hardwood forest subject to similar climatic conditions

Temporal variability in the ¹³C of foliage (¹³C_F), soil (¹³C_S) and ecosystem (¹³C_R) respired CO₂ was contrasted between a 17.2-m tall evenly aged loblolly pine forest and a 35-m tall unevenly aged mature second growth mixed broadleaf deciduous forest in North Carolina, USA, over a 2-year period. The two forests are located at the Duke Forest within a kilometer of each other and are subject to identical climate and have similar soil types. The ¹³C_F, collected just prior to dawn, was primarily controlled by the time-lagged vapor pressure deficit (VPD) in both stands; it was used for calculating the ratio of intercellular to ambient CO₂ (Ci/Ca). A remarkable similarity was observed in the relationship between Ci/Ca and time-lagged VPD in these two forests despite large differences in hydraulic characteristics. This similarity emerged as a result of physiological adjustments that compensated for differences in plant hydraulic characteristics, as predicted by a recently proposed equilibrium hypothesis, and has implications to ecophysiological models. We found that in the broadleaf forest, the ¹³C of forest floor CO₂ efflux dominated the ¹³C_R, while in the younger pine forest, the ¹³C of foliage respired CO₂ dominated ¹³C_R. This dependence resulted in a more variable ¹³C_R in the pine forest when compared to the broadleaf forest due to the larger photosynthetic contribution. Given the sensitivity of the atmospheric inversion models to ¹³C_R, the results demonstrate that these models could be improved by accounting for stand characteristics, in addition to previously recognized effects of moisture availability, when estimating ¹³C_R.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

External Ni<Superscript>2+</Superscript> and ENaC in A6 Cells: Na<Superscript>+</Superscript> Current Stimulation by Competition at a Binding Site for Amiloride and Na<Superscript>+</Superscript>

In cultured A6 monolayers from distal Xenopus kidney, external Ni²⁺ stimulated active Na⁺ uptake via the epithelial Na⁺ channel, ENaC. Transepithelial capacitance measurements ruled out exocytosis of ENaC-containing vesicles underlying the Ni²⁺ effect. Na⁺ current noise analysis was performed using the neutral Na⁺-channel blocker 6-chloro-3,5-diamino-pyrazine-2-carboxamide (CDPC) and amiloride. The analysis of CDPC-induced noise in terms of a three-state channel model revealed that Ni²⁺ elicits an increase in the number of open channels as well as in the spontaneous open probability. While Ni²⁺ had no influence on CDPC-blocker kinetics, the macroscopic and microscopic blocking kinetics of amiloride were affected. Ni²⁺ turned out to compete with amiloride for a putative binding site but not with CDPC. Moreover, external Na⁺—known to compete with amiloride and so producing the self-inhibition phenomenon—and Ni²⁺ exerted mutually exclusive analogous effects on amiloride kinetics. Na⁺ current kinetics revealed that Ni²⁺ prevents ENaC to be downregulated by self-inhibition. Co²⁺ behaved similarly to Ni²⁺, whereas Zn²⁺ did not. Attempts to disclose the chemical nature of the site reacting with Ni²⁺ suggested cysteine but not histidine as reaction partner.     

1-<Superscript>13</Superscript>C amino acid selective labeling in a <Superscript>2</Superscript>H<Superscript>15</Superscript>N background for NMR studies of large proteins

Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging protein systems. Here we present an approach where LBRT is achieved by adding protonated ¹⁴N amino acids that are ¹³C labeled at the carbonyl position to a medium for uniform deuteration and ¹⁵N labeling. This has three important benefits over conventional ¹⁵N LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid pairs connected by the labeling, and the amide proton of the residue following the ¹³C labeled amino acid is very sharp since its alpha position is deuterated, (2) the ¹³C label at the carbonyl position is less prone to scrambling than the ¹⁵N at the α-amino position, and (3) the peaks for the 1-¹³C labeled amino acids can be identified easily from the large intensity reduction in the ¹H-¹⁵N TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach is cost effective and has been successfully applied to proteins larger than 40 kDa. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of a Conserved Calmodulin-Binding Motif &;#x220A; the Sequence of F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase Inhibitor Protein

The natural inhibitor proteins IF₁ regulate mitochondrial F₀F₁ATPsynthase in a wide range of species. We characterized the interaction of CaM with purified bovine IF₁, two bovine IF₁ synthetic peptides, as well as two homologous proteins from yeast, namely IF₁ and STF₁. Fluorometric analyses showed that bovine and yeast inhibitors bind CaM with a 1:1 stoichiometry in the pH range between 5 and 8 and that CaM-IF₁ interaction is Ca²⁺-dependent. Bovine and yeast IF₁ have intermediate binding affinity for CaM, while the K_d (dissociation constant) of the STF₁-CaM interaction is slightly higher. Binding studies of CaM with bovine IF₁ synthetic peptides allowed us to identify bovine IF₁ sequence 33–42 as the putative CaM-binding region. Sequence alignment revealed that this region contains a hydrophobic motif for CaM binding, highly conserved in both yeast IF₁ and STF₁ sequences. In addition, the same region in bovine IF₁ has an IQ motif for CaM binding, conserved as an IQ-like motif in yeast IF₁ but not in STF₁. Based on the pH and Ca²⁺ dependence of IF₁ interaction with CaM, we suggest that the complex can be formed outside mitochondria, where CaM could regulate IF₁ trafficking or additional IF₁ roles not yet clarified.     

Enzymes involved in the anoxic utilization of phenyl methyl ethers by <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> DCB2 and <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> PCE-S

Phenyl methyl ethers are utilized by Desulfitobacterium hafniense DCB2 and Desulfitobacterium hafniense PCE-S; the methyl group derived from the O-demethylation of these substrates can be used as electron donor for anaerobic fumarate respiration or dehalorespiration. The activity of all enzymes involved in the oxidation of the methyl group to carbon dioxide via the acetyl-CoA pathway was detected in cell extracts of both strains. In addition, a carbon monoxide dehydrogenase activity could be detected. Activity staining of this enzyme indicated that the enzyme is a bifunctional CO dehydrogenase/acetyl-CoA synthase.     

A comparative theoretical study of the catalytic activities of Au<Stack><Subscript>2</Subscript><Superscript>-</Superscript></Stack> and AuAg<Superscript>-</Superscript> dimers for CO oxidation

The detailed mechanisms of catalytic CO oxidation over Au₂^- and AuAg^- dimers, which represent the simplest models for monometal Au and bimetallic Au-Ag nanoparticles, have been studied by performing density functional theory calculations. It is found that both Au₂^- and AuAg^- dimers catalyze the reaction according to the similar mono-center Eley–Rideal mechanism. The catalytic reaction is of the multi-channel and multi-step characteristic, which can proceed along four possible pathways via two or three elementary steps. In AuAg^-, the Au site is more active than the Ag site, and the calculated energy barrier values for the rate-determining step of the Au-site catalytic reaction are remarkably smaller than those for both the Ag-site catalytic reaction and the Au₂^- catalytic reaction. The better catalytic activity of bimetallic AuAg^- dimer is attributed to the synergistic effect between Au and Ag atom. The present results provide valuable information for understanding the higher catalytic activity of Au-Ag nanoparticles and nanoalloys for low-temperature CO oxidation than either pure metallic catalyst.     

Crystallographic evidence for a CO/CO<Subscript>2</Subscript> tunnel gating mechanism in the bifunctional carbon monoxide dehydrogenase/acetyl coenzyme A synthase from<Emphasis Type="Italic"> Moorella thermoacetica</Emphasis>

Acetyl coenzyme A synthase (ACS) acts in concert with carbon monoxide dehydrogenase (CODH) to catalyze the formation of acetyl-coenzyme A from CO₂-derived CO and CH₃⁺ molecules. Recent crystal structures have shown that the three globular domains constituting the ACS subunit may be arranged in either a closed or an open conformation. A long hydrophobic tunnel network allows diffusion of CO between the CODH and the ACS active sites in the closed form, but it is blocked in the open form. On the other hand, the active site of ACS is only accessible for coenzyme A and the methyl donating protein in the open domain conformation. Although several metal compositions have been observed for this active site, present consensus is that it consists of a Ni-Ni-[Fe₄S₄] cluster. The observed conformational changes of ACS and the resulting different substrate accessibilities of the catalytic central nickel are reviewed here in the context of a putative CO₂/CO tunnel gating mechanism.     

Performance of a generalist grasshopper on a C<Subscript>3</Subscript> and a C<Subscript>4</Subscript> grass: compensation for the effects of elevated CO<Subscript>2</Subscript> on plant nutritional quality

The increasing CO₂ concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C₃ than C₄ plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C₃ plants. These hypotheses were tested by growing the grasses Lolium multiflorum C₃) and Bouteloua curtipendula C₄) at ambient (370 ppm) and elevated (740 ppm) CO₂ levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO₂ treatment combinations. As expected, the nutritional quality of the C₃ grass declined to a greater extent than did that of the C₄ grass at elevated CO₂; protein levels declined in the C₃ grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C₃ grass grown under elevated CO₂. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C₃ grass under elevated CO₂. Consumption rates of the C₃ and C₄ grasses were also similar, demonstrating a lack of compensatory feeding on the C₄ grass. We also examined the relative efficiencies of nutrient utilization from a C₃ and C₄ grass by M. sanguinipes to test the basis for the C₄ plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C₄ grass than from the C₃ grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C₃ grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C₃ grass and the better growth performance of M. sanguinipes on this grass at both CO₂ levels, we conclude that C₃ grasses are likely to remain better host plants than C₄ grasses in future CO₂ conditions.     

Physiological characteristics of the primitive CO<Subscript>2</Subscript> concentrating mechanism in PEPC transgenic rice

The relationship between carbon assimilation and high-level expression of the maize PEPC in PEPC transgenic rice was studied by comparison to that in the untransformed rice, japonica kitaake. Stomatal conductance and photosynthetic rates in PEPC transgenic rice were higher than those of untransformed rice, but the increase of stomatal conductance had no statistical correlation with that of photosynthetic rate. Under high levels of light intensity, the protein contents of PEPC and CA were increased significantly. Therefore the photosynthetic capacity was increased greatly (50%) with atmospheric CO2 supply. While CO2 release in leaf was reduced and the compensation point was lowered correspondingly under CO2 free conditions. Treatment of the rice with the PEPC-specific inhibitor DCDP showed that overexpression of PEPC and enhancement of carbon assimilation were related to the stability of Fv/Fm. Labeling with 14CO2 for 20 s showed more 14C was distributed to C4 primary photosynthate asperate in PEPC transgenic rice, suggesting that there exists a limiting C4 photosynthetic mechanism in leaves. These results suggest that the primitive CO2 concentrating mechanism found in rice could be reproduced through metabolic engineering, and shed light on the physiological basis for transgenic breeding with high photosynthetic efficiency.     

Stress-protective activity of the CH<Subscript>3</Subscript>CO-Lys-Lys-Arg-Arg-NH<Subscript>2</Subscript> synthetic peptide (protectin)

The CH₃CO-Lys-Lys-Arg-Arg-NH₂ peptide (the author has named it protectin) was synthesized, and its activity was studied during different stress actions. Protectin was found to normalize the content of corticosterone and adrenalin in adrenal glands and blood after its intranasal administration to rats one day before a cold or heat shock, or hypobaric hypoxia at doses of 1–10 μg/animal and after its intravenous administration just after acute hemorrhage at doses of 0.5–2 μg/animal. The intranasal administration of protectin at doses of 1–10 μg/rat one day before the heat or cold shock was also shown to prevent a change in the content of free histamine and the activity of diamine oxidase in myocardium, which was induced by the dramatic change in the activity of the enzyme after the temperature actions.     

Intramolecular G-quadruplexes formed by d(GT)<Subscript>12</Subscript> microsatellite sequence in the presence of K<Superscript>+</Superscript>

Polymorphic d(GT) _n microsatellite sequences are known to dramatically affect the regulation of gene expression. CD spectroscopy, UV melting, fluorescence polarization of ethidium bromide (EtBr), and FRET allowed the detection of a new G-quartet fold formed by the d(GT)₁₂ oligonucleotide in 0.01 M Na-phosphate (pH 8.0) in the presence of 0.1 M KCl. The monomolecular type of the structure was verified by measuring the rotational relaxation time (ρ = 28 ± 0.5 ns) of the EtBr: d(GT)₁₂ complex. CD spectra supported the G-quartet formation. FRET was used to estimate the distance between intercalated EtBr and FITC covalently attached to the 5′ end of d(GT)₁₂ (R ≤ 17 Å). The experimental data agree with the self-folding of d(GT)₁₂ in a G-quartet structure.     

Differential gene expression during apoptosis induced by a serum factor: Role of mitochondrial F<Subscript>0</Subscript>-F<Subscript>1</Subscript> ATP synthase complex

The number of genes that are up regulated or down regulated during apoptosis is large and still increasing. In an attempt to characterize differential gene expression during serum factor induced apoptosis in AK-5 cells (a rat histiocytoma), we found subunit 6 and subunit 8 of the transmembrane proton channel and subunit alpha of the catalytic core of the mitochondrial F₀-F₁ ATP synthase complex to be up regulated during apoptosis. The increase in the expression levels of these subunits was concomitant with a transient increase in the intracellular ATP levels, suggesting that the increase in cellular ATP content is a result of the increase in the expression of ATP synthase subunits' gene and de novo protein synthesis. Depleting the cellular ATP levels with oligomycin inhibited apoptosis significantly, pointing to the requirement of ATP during apoptosis. Caspase 1 and caspase 3 activity and the loss of mitochondrial membrane potential were also inhibited by oligomycin during apoptosis in these cells, suggesting that the oligomycin induced inhibition of apoptosis could be due to inhibition of caspase activity and inhibition of mitochondrial depolarization. However, cytochrome C release during apoptosis was found to be completely independent of intracellular ATP content. Besides the ATP synthase complex genes, other mitochondrial genes like cytochrome C oxidase subunit II and III also showed elevated levels of expression during apoptosis. This kind of a mitochondrial gene expression profile suggests that in AK-5 cells, these genes are upregulated in a time-linked manner to ensure sufficient intracellular ATP levels and an efficient functioning of the mitochondrial respiratory chain for successful completion of the apoptotic pathway.     

The contribution of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> plants to the carbon cycle of a tallgrass prairie: an isotopic approach

The photosynthetic pathway composition (C₃:C₄ mixture) of an ecosystem is an important controller of carbon exchanges and surface energy flux partitioning, and therefore represents a fundamental ecophysiological distinction. To assess photosynthetic mixtures at a tallgrass prairie pasture in Oklahoma, we collected nighttime above-canopy air samples along concentration and isotopic gradients throughout the 1999 and 2000 growing seasons. We analyzed these samples for their CO₂ concentration and carbon isotopic composition and calculated C₃:C₄ proportions with a two-source mixing model. In 1999, the C₄ percentage increased from 38% in spring (late April) to 86% in early fall (mid-September). The C₄ percentages inferred from ecosystem respiration measurements in 2000 indicate a smaller shift, from 67% in spring (early May) to 77% in mid-summer (late July). We also sampled daytime CO₂ concentration and carbon isotope gradients above the canopy to determine ecosystem discrimination against ¹³CO₂ during net uptake. These discrimination values were always lower than corresponding nighttime ecosystem respiration isotopic signatures would suggest. After accounting for the isotopic disequilibria between respiration and photosynthesis resulting from seasonal variations in the C₃:C₄ mixture, we estimated canopy photosynthetic discrimination. The C₄ percentage calculated from this approach agrees with the percentage determined from nighttime respiration for sampling periods in both growing seasons. Isotopic imbalances between photosynthesis and respiration are likely to be common in mixed C₃:C₄ ecosystems and must be considered when using daytime isotopic measurements to constrain ecosystem physiology. Given the global extent of such ecosystems, isotopic imbalances likely contribute to global variations in the carbon isotopic composition of atmospheric CO₂.     

BODIPY for photodynamic therapy applications: computational study of the effect of bromine substitution on <Superscript>1</Superscript>O<Subscript>2</Subscript> photosensitization

Density functional theory and its time-dependent extension (DFT, TDDFT) were employed to establish the feasibility of using a series of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs) in photodynamic therapy. Their absorption electronic spectra, singlet–triplet energy gaps, and spin–orbit matrix elements were computed and are discussed here. The effects of bromine substitution on the photophysical properties of BODIPY were elucidated. The investigated compounds were found to possess different excited triplet states that lie below the energy of the bright excited singlet state (S₁ or S₂), depending on the positions occupied by the bromine atoms. The computed spin–orbit matrix elements for the radiationless intersystem crossing S_n?→ ?T_m and the relative singlet–triplet energy gaps allowed the prediction of plausible nonradiative decay pathways for the production of singlet excited molecular oxygen, the key cytotoxic agent in photodynamic therapy.

Open image in new window

Graphical Abstract The photophysical properties affected by the presence of bromine atoms in different positions of a BODIPY core have been here elucidated. In particular it has been found that SOC values strongly depend on the position of heavy atoms into the BODIPY core, suggesting positions 1 and 7 as the best ones to enhance the ISC kinetics

     

Vasotocin has the potential to inhibit basolateral Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-pump current across isolated skin of tree frog in vitro<Emphasis Type="Italic">,</Emphasis> via its V<Subscript>2</Subscript>-type receptor/cAMP pathway

Adult frog skin transports Na⁺ from the apical to the basolateral side across the skin. Antidiuretic hormone (ADH) is involved in the regulation of Na⁺ transport in both mammals and amphibians. We investigated the effect of arginine vasotocin (AVT), the ADH of amphibians, on the short-circuit current (SCC) across intact skin and on the basolateral Na⁺/K⁺-pump current across apically nystatin-permeabilized skin of the tree frog, Hyla japonica, in which the V₂-type ADH receptor is expressed in vitro. In intact skin, 1 pM AVT had no effect on the SCC, but 10 nM AVT was sufficient to stimulate the SCC since 10 nM and 1 μM of AVT increased the SCC 3.2- and 3.4-fold, respectively (P > 0.9). However, in permeabilized skin, AVT (1 μM) decreased the Na⁺/K⁺-pump current to 0.79 times vehicle control. Similarly, 500 μM of 8Br-cAMP increased the SCC 3.2-fold, yet 1 mM of 8Br-cAMP decreased the Na⁺/K⁺-pump current to 0.76 times vehicle control. Arachidonic acid (10⁻⁵ M) tended to decrease the Na⁺/K⁺-pump current. To judge from these in vitro experiments, AVT has the potential to inhibit the basolateral Na⁺/K⁺-pump current via the V₂-type receptor/cAMP pathway in the skin of the tree frog.     

Manipulating the Length of the <Emphasis Type="Italic">b</Emphasis> Subunit F<Subscript>1</Subscript> Binding Domain in F<Subscript>1</Subscript>F<Subscript>0</Subscript> ATP Synthase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The peripheral stalk of F₁F₀ ATP synthase is composed of a parallel homodimer of b subunits that extends across the cytoplasmic membrane in F₀ to the top of the F₁ sector. The stalk serves as the stator necessary for holding F₁ against movement of the rotor. A series of insertions and deletions have been engineered into the hydrophilic domain that interacts with F₁. Only the hydrophobic segment from {val-121} to {ala-132} and the extreme carboxyl terminus proved to be highly sensitive to mutation. Deletions in either site apparently abolished enzyme function as a result of defects is assembly of the F₁F₀ complex. Other mutations manipulating the length of the sequence between these two areas had only limited effects on enzyme function. Expression of a b subunit with insertions with as few as two amino acids into the hydrophobic segment also resulted in loss of F₁F₀ ATP synthase. However, a fully defective b subunit with seven additional amino acids could be stabilized in a heterodimeric peripheral stalk within a functional F₁F₀ complex by a normal b subunit.