A valid quantitative histochemical technique for assaying cytochrome c oxidase in single cells

A quantitative histochemical method for assaying cytochrome c oxidase (COX) has been validated with two new findings concerning the optimal tissue thickness and a suitable substrate. The kinetics of a COX-catalysed reaction coupled to the oxidation of diaminobenzidine (DAB) were followed at 37 degrees C in single muscle fibres in unfixed sections of mouse gastrocnemius using a real-time image analysis system. The optimum composition of the substrate medium for the reaction was 0.1 mM reduced cytochrome c, 4 mM DAB, 2% dimethylsulphoxide, 2% polyvinyl alcohol and 0.1 mM HEPES buffer, final pH 7.5. The absorbances at 451 nm of the final reaction products, DAB polymer oxides, deposited in the intermyofibrillar mitochondria increased linearly as a function of incubation time for at least 80 s after the start of incubation. The initial velocities (v(i)) of the COX reaction calculated from the gradients of the linear regression best fits for times between 40 and 60 s were reproducible. The v(i) determined in single muscle fibres at a saturated concentration of cytochrome c (0.1 mM) were proportional to section thickness for thicknesses less than 3 microns, but they decreased exponentially when the thickness was greater than 4 microns. Thus, for the quantitative assay, unfixed sections 3 microns thick must be used. The Michaelis constants (Km) determined for commercial cytochrome c in the range of 20-26 microM for COX in three types of skeletal muscle fibres of mouse gastrocnemius were higher than the corresponding in situ Km (12-13 microM) for reduced cytochrome c. However, the Km values for commercial cytochrome c were in good agreement with the value previously determined with homogenates of rat hind limb muscle. Therefore, reduced cytochrome c is a more suitable substrate for the kinetic study and assay of COX in situ.     

Electrochemical analysis of the effect of Ca on cardiolipin–cytochrome c interaction

Mitochondrial Ca²⁺ has been considered a trigger for the release of cytochrome c, which is a critical and early event in the induction of cell apoptosis, although the molecular mechanism underlying this effect is still not fully understood. Here we investigate the interaction between cytochrome c and cardiolipin and the effect of Ca²⁺ on this interaction using electrochemical methods. Experimental results revealed that modification of cardiolipin onto the surface of a pyrolytic graphite electrode could lead to a rapid direct electron transfer of cytochrome c through the electrostatic interaction between the protein and the cardiolipin. Addition of Ca²⁺ to the test solution containing cytochrome c could cause the decrease of the redox peaks of the protein, and the peaks could be recovered when Ca²⁺ was chelated by ethylenediaminetetraacetate. The cardiolipin–cytochrome c interaction and the Ca²⁺ effect were also investigated with the variation of the charges of lipids, buffer solutions, reaction time, and valencies of cations for comparison.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

Biochemical and biophysical studies on cytochrome c oxidase. XVII. An epr study of the photodissociation of cytochrome a32+ · CO

1. The photodissociation reaction of the cytochrome c oxidase-CO compound was studied by EPR at 15 °K. Illumination with white light at both room and liquid N₂ temperatures of the partially reduced cytochrome c oxidase (2 electrons per 4 metals) in the presence of CO, causes the appearance of a rhombic (g_x = 6.60, g_y = 5.37) high-spin heme signal.This signal disappears completely upon darkening of the sample and reappears upon illumination at room temperature; accordingly the photolytic process is reversible. Under these conditions, no great changes in the intensities are observed, neither of the copper signal at g = 2, nor of the low-spin heme signal at g = 3, 2.2 and 1.5.2. In the presence of ferricyanide (2 mM) and CO, both the low-spin heme signal (g = 3.0, 2.2 and 1.5) and the copper signal of the partially reduced enzyme have intensities about equal to those of the completely oxidized enzyme in the absence of CO. Upon illumination of the carboxy-cytochrome c oxidase in the presence of ferricyanide, it was found that the rhombic high-spin heme signal appears without affecting appreciably the copper of low-spin heme signals. Thus, in the presence of ferricyanide the EPR-detectable paramagnetism of the illuminated carboxy-cytochrome c oxidase is higher than in the untreated oxidized enzyme.3. The membrane-bound cytochrome c oxidase reduced with NADH in the presence of CO and subsequently oxidized with ferricyanide shows a similar rhombic high-spin heme signal (g_x = 6.62, g_y = 5.29) upon illumination at room temperature. This signal disappears completely upon darkening and reappears upon illumination at room temperature.     

The effect of lysozyme amyloid fibrils on cytochrome c–lipid interactions

Protein polymerization into ordered fibrillar structures (amyloid fibrils) is currently associated with a range of pathological conditions. Recent studies clearly indicate that amyloid cytotoxicity is provoked by a continuum of cross-β-sheet aggregates including mature fibrils. In view of the possible diversity of cytotoxicity mechanisms, the present study addressed the question of whether protein conversion into amyloid fibrils can modify its competitive membrane adsorption behavior. Using a combination of resonance energy transfer, dynamic light scattering and fluorescence quenching techniques, the competitive binding of either monomeric or polymerized lysozyme, and cytochrome c to the model lipid membranes composed of phosphatidylcholine mixtures with varying proportions of phosphatidylglycerol, phosphatidylserine or cardiolipin has been studied. The ability of fibrillar lysozyme to induce dissociation of cytochrome c from the membrane binding sites proved to be markedly stronger than that of its monomeric counterpart, with desorption process displaying cooperativity features upon increasing the charge of lipid bilayer. The decreased efficiency of tryptophan fluorescence quenching by acrylamide and short-wavelength shift of emission maximum observed upon membrane binding of lysozyme fibrils were rationalized in terms of fluorophore transfer into interfacial bilayer region. It is hypothesized that electrostatic interactions play predominant role in determining the lipid-associating and competitive abilities of fibrillar lysozyme.     

M?ssbauer study of a bacterial cytochrome oxidase: cytochrome c1aa3 from Thermus thermophilus

Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase. We have studied 87Fe-enriched samples with M?ssbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide. The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes. The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with M?ssbauer parameters quite similar to those of deoxymyoglobin. Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes. The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state. We have examined the oxidized enzyme from two different preparations. Both had good activity and identical optical and EPR spectra. The M?ssbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations. Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme.     

Yeast cytochrome c oxidase: A model system to study mitochondrial forms of the haem–copper oxidase superfamily

The known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. The structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit I. Yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Ferredoxin:NADP+ oxidoreductase bound to cytochrome b₆f complex is active in plastoquinone reduction: implications for cyclic electron transport

In this study, we have compared three isolation methods of cytochrome b₆f complex, obtained from spinach (Spinacia oleracea), differing in the preservation of the cytochrome b₆f‐associated ferredoxin:NADP⁺ oxidoreductase (FNR). Although the complexes isolated by all the methods showed the presence of the FNR peptide(s), when incorporated into liposome membranes, the NADPH‐PQ (plastoquinone) oxidoreductase activity was not detected for the cytochrome b₆f complex isolated with the original method including a NaBr wash. Some activity was found for the complex isolated with the omission of the wash, but the highest activity was detected for the complex isolated with the use of digitonin. The reaction rate of PQ reduction of the investigated complexes in liposomes was not significantly influenced by the addition of free FNR or ferredoxin. The reaction was inhibited by about 60% in the presence of 2 µM 2‐n‐nonyl‐4‐hydroxyquinoline N‐oxide, an inhibitor of the cytochrome b₆f complex at the Q_i site, while it was not affected by triphenyltin or isobutyl cyanide that interacts with the recently identified heme c_i. The obtained data indicate that FNR associated with the cytochrome b₆f complex can participate in the cyclic electron transport as PSI‐PQ or NADPH‐PQ oxidoreductase. Moreover, we have shown that PQ can be non‐enzymatically reduced by ascorbate in liposomes and this reaction might be involved in non‐photochemical reduction pathways of the PQ‐pool in chloroplasts.     

Comparison of DNA extraction methods for PCR amplification of mitochondrial cytochrome c oxidase subunit II (COII) DNA from primate fecal samples

Mitochondrial COII DNA was amplified by PCR from total DNA extracted from field collected primate fecal samples (n=24) which had been stored without refrigeration for over 30 days. High molecular weight DNA total DNA was obtained from samples stored in 70% (v/v) ethanol, SDS lysis buffer (LB) and guanidine isothiocyanate buffer (GTB) than from samples stored in 10% formalin. Fecal DNA quality and COII amplification varied according to storage solution (formalin, ethanol, LB and GTB), extraction method (LB-based and GTB-based) and primate species (chimpanzee, baboon, human). It is recommended that fecal samples be collected in LB for DNA analysis. However, GTB-based protocols are suitable when total RNA is needed for epidemiological studies of viral diseases or gene expression analysis.     

Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence

In addition to its electron transfer activity, cytochrome c is now known to trigger apoptosis via peroxidase activity. This new function is related to a structural modification of the cytochrome upon association with anionic lipids, particularly cardiolipin present in the mitochondrial membrane. However, the exact nature of the non-native state induced by this interaction remains an active subject of debate. In this work, using human cytochromes c (native and two single-histidine mutants and the corresponding double mutant) and micelles as a hydrophobic medium, we succeeded, through UV–visible spectroscopy, circular dichroism spectroscopy and NMR spectroscopy, in fully characterizing the nature of the sixth ligand replacing the native methionine. Furthermore, careful pH titrations permitted the identification of the amino acids involved in the iron binding over a range of pH values. Replacement of the methionine by lysine was only observed at pH above 8.5, whereas histidine binding is dependent on both pH and micelle concentration. The pH variation range for histidine protonation is relatively narrow and is consistent with the mitochondrial intermembrane pH changes occurring during apoptosis. These results allow us to rule out lysine as the sixth ligand at pH values close to neutrality and reinforce the role of histidines (preferentially His33 vs. His26) as the main candidate to replace methionine in the non-native cytochrome c. Finally, on the basis of these results and molecular dynamics simulations, we propose a 3D model for non-native cytochrome c in a micellar environment.     

Evidence for two H2O2-binding sites in ferric cytochrome c oxidase. Indication to the O-cycle?

H2O2 addition to the oxidized cytochrome c oxidase reconstituted in liposomes brings about a red shift of the Soret band of the enzyme and an increased absorption in the visible region with two distinct peaks at approximately 570 and 605 nm. Throughout pH range 6-8.5, the spectral changes at 570 nm and in the Soret band titrate with very similar pH-independent Kd values of 2-3 microM. At the same time, Kd of the peroxide complex measured at 605 nm increases markedly with increased H+ activity reaching the value of 18 +/- 2 microM at pH 6.0. This finding may indicate the presence of two different H2O2-binding sites in the enzyme with different affinity for the ligand at acid pH. The Soret and 570 nm band effects are suggested to report H2O2 coordination to heme iron of alpha 3, whereas the maximum at 605 nm could arise from H2O2 binding to Cu alpha 3 followed by the enzyme transition into the 'pulsed' (or '420/605') conformation. Possible implication of the two H2O2-binding sites for the cytochrome oxidase redox and proton-pumping mechanisms are discussed.     

The accelerated evolution of human cytochrome c oxidase – Selection for reduced rate and proton pumping efficiency?

The cytochrome c oxidase complex, complex VI (CIV), catalyzes the terminal step of the mitochondrial electron transport chain where the reduction of oxygen to water by cytochrome c is coupled to the generation of a protonmotive force that drive the synthesis of ATP. CIV evolution was greatly accelerated in humans and other anthropoid primates and appears to be driven by adaptive selection. However, it is not known if there are significant functional differences between the anthropoid primates CIV, and other mammals. Comparison of the high-resolution structures of bovine CIV, mouse CIV and human CIV shows structural differences that are associated with anthropoid-specific substitutions. Here I examine the possible effects of these substitutions in four CIV peptides that are known to affect proton pumping: the mtDNA-coded subunits I, II and III, and the nuclear-encoded subunit VIa2. I conclude that many of the anthropoid-specific substitutions could be expected to modulate the rate and/or the efficiency of proton pumping. These results are compatible with the previously proposed hypothesis that the accelerated evolution of CIV in anthropoid primates is driven by selection pressure to lower the mitochondrial protonmotive force and thus decrease the rate of superoxide generation by mitochondria.     

Antioxidative effect of α-tocopherol incorporation into lecithin liposomes on ascorbic acid-Fe2+-induced lipid peroxidation

The antioxidative effect of α-tocopherol incorporated into lecithin liposomes was studied. Lipid peroxidation of liposome membranes, assayed as malondialdehyde production, was catalyzed by ascorbic acid and Fe²⁺. The peroxidation reaction, which did not involve the formation of singlet oxygen, superoxide, hydrogen peroxide, or a hydroxyl radical, was inhibited by α-tocopherol and a model compound of α-tocopherol, 2,2,5,7,8-pentamethyl-6-hydroxy-chroman (TMC), but not by phytol, α-tocopherylquinone, or α-tocopheryl acetate. One mole of α-tocopherol completely prevented peroxidation of about 100 moles of polyunsaturated fatty acid. Decrease in membrane fluidity by lipid peroxidation, estimated as increase of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in the membrane, was also inhibited by α-tocopherol and TMC, reflecting their antioxidant functions. Cholesterol did not act as an antioxidant, even when incorporated in large amount into the liposome membranes, but it increased the antioxidative efficiency of α-tocopherol. When a mixture of liposomes with and without α-tocopherol was incubated with Fe²⁺ and ascorbic acid, α-tocopherol did not protect the liposomes not containing α-tocopherol from peroxidation. However, preincubation of the mixture, or addition of Triton X-100 allowed the α-tocopherol to prevent peroxidation of the liposomes not containing α-tocopherol. In contrast, in similar experiments, liposomes containing TMC prevented peroxidation of those without TMC without preincubation. Tocopherol in an amount so small as to exhibit only a slight antioxidative effect was oxidized when incorporated in egg lecithin liposomes, but it mostly remained unoxidized when incorporated in dipalmitoyllecithin liposomes, indicating that oxygen activated by ascorbic acid-Fe²⁺ does not oxidize α-tocopherol directly. Thus, decomposition of α-tocopherol may be caused by its interaction with peroxy and/or alkoxyl radicals generated in the process of lipid peroxidation catalyzed by Fe²⁺ and ascorbic acid.     

Calculated proton uptake on anaerobic reduction of cytochrome C oxidase: is the reaction electroneutral?

Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O(2). Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a(3) and Cu(B). If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK(a)s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu(B) shifts its pK(a) from below 7 to above 7 and so picks up a proton when heme a(3) and Cu(B) are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a(3) all have pK(a)s above 7 in all redox states, although they have only 1.6-3.5 DeltapK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK(a)s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu(A), heme a, heme a(3), and Cu(B) shows approximately 2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E(m)) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E(m) of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E(m) is independent of the BNC redox state.     

Use of 3,3′-diaminobenzidine as a biochemical electron donor for studies on terminal cytochrome oxidase activity inAzotobacter vinelandii

Azotobacter vinelandii cells readily oxidize the dye 3,3′-diaminobenzidine (DAB), which has been previously used as an electron donor for studies on the mitochondrial cytochromec oxidase reaction. The DAB oxidase activity inA. vinelandii cells was 10-fold lower than that noted for theN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase reaction, which is commonly used to measure terminal oxidase activity both in bacteria and mitochondria. Analyses of cell-free extracts show that DAB oxidase activity is concentrated almost exclusively in theA. vinelandii membrane fractions, most notably in the “R₃” electron transport particle (ETP). Oxidation studies, which employed both whole cells and the ETP fraction, show DAB oxidase activity to be markedly sensitive to KCN, NaN₃, and NH₂OH. A manometric assay system was developed which readily measured DAB oxidase activity in bacteria. Preliminary studies indicate that ascorbate-DAB oxidation inAzotobacter vinelandii measures terminal cytochrome oxidase activity in a manner similar to the TMPD oxidase reaction.     

The effect of ethanol on the formation of N 2-ethylidene-dG adducts in mice: implications for alcohol-related carcinogenicity of the oral cavity and esophagus

The present study aimed to experimentally confirm that long-term alcohol drinking causes a high risk of oral and esophageal cancer in aldehyde dehydrogenase 2 (ALDH2)-deficient individuals. Aldh2 knockout mice, an animal model of ALDH2-deficiency, were treated with 8% ethanol for 14 months. Levels of acetaldehyde-derived DNA adducts were increased in esophagus, tongue and submandibular gland. Our finding that a lack of Aldh2 leads to more DNA damage after chronic ethanol treatment in mice supports epidemiological findings on the carcinogenicity of alcohol in ALDH2-deficient individuals who drink chronically.     

Reversal of cyanide inhibition of cytochrome c oxidase by the auxiliary substrate nitric oxide: an endogenous antidote to cyanide poisoning?

Nitric oxide (NO) is shown to overcome the cyanide inhibition of cytochrome c oxidase in the presence of excess ferrocytochrome c and oxygen. Addition of NO to the partially reduced cyanide-inhibited form of the bovine enzyme is shown by electron paramagnetic resonance spectroscopy to result in substitution of cyanide at ferriheme a3 by NO with reduction of the heme. The resulting nitrosylferroheme a3 is a 5-coordinate structure, the proximal bond to histidine having been broken. NO does not simply act as a reversibly bound competitive inhibitor but is an auxiliary substrate consumed in a catalytic cycle along with ferrocytochrome c and oxygen. The implications of this observation with regard to estimates of steady-state NO levels in vivo is discussed. Given the multiple sources of NO available to mitochondria, the present results appear to explain in part some of the curious biomedical observations reported by other laboratories; for example, the kidneys of cyanide poisoning victims surprisingly exhibit no significant irreversible damage, and lethal doses of potassium cyanide are able to inhibit cytochrome c oxidase activity by only approximately 50% in brain mitochondria.     

Molecular evolution of cytochrome c oxidase subunit I in primates: is there coevolution between mitochondrial and nuclear genomes?

Phylogenetic analyses carried out on cytochrome c oxidase (COX) subunit I mitochondrial genes from 14 primates representing the major branches of the order and four outgroup nonprimate eutherians revealed that transversions and amino acid replacements (i.e., the more slowly occurring sequence changes) contained lower levels of homoplasy and thus provided more accurate information on cladistic relationships than transitions (i.e., the more rapidly occurring sequence changes). Several amino acids, each with a high likelihood of functionality involving the binding of cytochrome c or interaction with COX VIII, have changed in Anthropoidea, the primate suborder grouping New World monkey, Old World monkey, ape, and human lineages. They are conserved in other mammalian lineages and in nonanthropoid primates. Maximum-likelihood ancestral COX I nucleotide sequences were determined utilizing a near most parsimonious branching arrangement for the primate sequences that was consistent with previously hypothesized primate cladistic relationships based on larger and more diverse data sets. Relative rate tests of COX I mitochondrial sequences showed an elevated nonsynonymous (N) substitution rate for anthropoid-nonanthropoid comparisons. This finding for the largest mitochondrial (mt) DNA-encoded subunit is consistent with previous observations of elevated nonsynonymous substitution/synonymous substitution (S) rates in primates for mt-encoded COX II and for the nuclear-encoded COX IV and COX VIIa-H. Other COX-related proteins, including cytochrome c and cytochrome b, also show elevated amino acid replacement rates or N/S during similar time frames, suggesting that this group of interacting genes is likely to have coevolved during primate evolution.     

Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase

Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo₃ oxidase. The optimized protocol for expression and purification yields ∼5 mg of the enzymatically active, uniformly ¹³C,¹⁵N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The ¹³C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo₃ oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly ¹³C,¹⁵N-labeled membrane protein. Heather L. Frericks and Lai Lai Yap contributed equally to this work.