Subunits VIIa,b,c of human cytochrome c oxidase. Identification of both 'heart-type' and 'liver-type' isoforms of subunit VIIa in human heart.

The N-terminal amino acid sequences and the electrophoretic mobilities of the subunits VIIa, VIIb and VIIc of cytochrome c oxidase purified from human heart were investigated and compared with those from human skeletal muscle and from bovine heart. In purified human heart cytochrome c oxidase, both so-called 'heart-type' and 'liver-type' isoforms of subunit VIIa were found. The first 30 residues of the N-terminal amino acid sequences of these 'heart-type' and 'liver-type' subunits VIIa showed nine differences. The two isoforms of subunit VIIa in human heart were present in almost equal amounts, in contrast to the situation in skeletal muscle, where the 'heart-type' subunit VIIa was predominant. Therefore, our results imply that in human heart a cytochrome c oxidase isoform pattern is present that differs from that found in skeletal muscle. Subunits VIIb and VIIc purified from human heart oxidase proved to be very similar to their bovine heart counterparts. Our direct demonstration of the presence of subunit VIIb, the sequence of which has only recently been identified in the bovine heart enzyme, suggests that human cytochrome c oxidase also contains 13 subunits. We found no evidence for the presence of different isoforms of subunit VIIc in cytochrome c oxidase from human heart and skeletal muscle. We observed clear differences in the electrophoretic mobility of the subunits VIIa,b,c between bovine and human cytochrome c oxidase. On Tricine/glycerol/SDS/polyacrylamide gels the 'heart-type' and 'liver-type' subunits VIIa present in human heart cytochrome c oxidase migrated with almost the same electrophoretic mobility. Subunit VIIb migrated only slightly faster than subunit VIIa, whereas VIIc proved to have the highest electrophoretic mobility on Tricine/SDS/glycerol/polyacrylamide gels. Our findings may have implications for the elucidation of certain tissue-specific cytochrome c oxidase deficiencies in man.     

Structure of the cytochrome c oxidase complex of rat liver. 2. Topological orientation of polypeptides in the membrane as studied by proteolytic digestion and immunoblotting

The orientation of the thirteen polypeptides of rat-liver cytochrome c oxidase in the inner mitochondrial membrane was studied by proteolytic digestion of mitoplasts and sonicated particles. After separation by sodium dodecylsulfate gel electrophoresis proteins were transferred on nitrocellulose, and individual polypeptides were identified by incubation with polypeptide-specific antisera, followed by fluorescein-isothiocyanate-conjugated protein A. The three catalytic polypeptides I-III and seven nuclear coded polypeptides (IV, Vb, VIa, VIc, VIIa, VIIb and VIII) were found accessible to proteases from the cytoplasmic phase. Polypeptides II, IV, Va, Vb and VIa were accessible from the matrix phase, indicating a transmembraneous orientation of polypeptides II, IV, Vb and VIa. Together with data on cross-linking and on cytochrome-c-protected labeling of polypeptides, a model of the cytochrome c oxidase complex was developed. It is suggested that the cytochrome c binding site on polypeptide II is surrounded by several nuclear-coded polypeptides, which may modulate the affinity of the enzyme towards cytochrome c.     

Tissue-specific and species-specific distribution of -SH groups in cytochrome c oxidase subunits

Cytochrome c oxidase was isolated from pig, bovine, rat and human tissues including liver, heart, diaphragm and kidney. The native and the sodium-dodecyl-sulfate (SDS)-dissociated enzymes were labelled under optimal conditions with N-ethyl-[2,3-14C]maleimide before and after reduction with dithiothreitol, separated into 13 subunits by SDS gel electrophoresis and the radioactive bands were visualized by fluorography. In some cases the radioactive bands were cut out and counted. All isozymes were labelled in subunits I, III, Va and VIIb, and in subunit II after reduction. Labelling of subunit Vb was equivocal, and in no case were subunits IV and VIc labelled. All other subunits were labelled tissue-specifically and/or species-specifically. No differences were found between labelling of the native and SDS-dissociated enzyme. By relating the molar amount of bound N-ethylmaleimide to the known amount of cysteines in subunits of bovine heart cytochrome c oxidase, the percentage of -SH group reactivity was calculated. Only the cysteine of subunit Va was found to be 100% reactive. The distinct and different reactivity of subunit VIIb as compared to subunits VIIa and VIIc clearly establishes this polypeptide as an independent subunit of mammalian cytochrome c oxidase.     

Cross reactivity of monoclonal antibodies and cDNA hybridization suggest evolutionary relationships between cytochrome c oxidase subunits VIa and VIc and between VIIa and VIIb.

Monoclonal antibodies to subunits of bovine heart cytochrome c oxidase were prepared by immunizing mice with the isolated enzyme. The majority of antibody-producing cell lines were found to react with two different subunits of similar molecular mass, as shown by Western blotting and ELISA titrations with the HPLC-purified subunits. The affinities of the monoclonal antibodies to the subunits were determined by ELISA titrations with increasing concentrations of NH4SCN. Two monoclonal antibodies with a low affinity to subunit VIa had a high affinity to subunit VIc, whereas two other antibodies showed the same affinity to subunits VIIa and VIIb. The same affinity of monoclonal antibodies suggested an evolutionary relationship of subunits VIIa and VIIb, which was further supported by reactivity of these antibodies to subunits VIIa and VIIb of cytochrome c oxidase from different species and tissues. Also the evolutionary relationship between subunit VIa and VIc was shown by hybridization at low stringency of cDNAs for rat cytochrome c oxidase subunits VIc and VIa-h (heart-type), after amplification by the polymerase chain reaction, with a probe of VIa-l (liver-type).     

Specific modification of two tryptophans within the nuclear-encoded subunits of bovine cytochrome c oxidase by hydrogen peroxide

Hydrogen peroxide does more than react with the binuclear center of oxidized bovine cytochrome c oxidase and generate the well-characterized "peroxy" and "ferryl" forms. Hydrogen peroxide also inactivates detergent-solubilized cytochrome c oxidase in a time- and concentration-dependent manner. There is a 70-80% decrease of electron-transport activity, peroxidation of bound cardiolipin, modification of two nuclear-encoded subunits (IV and VIIc), and dissociation of approximately 60% of subunits VIa and VIIa. Modification of subunit VIIc and dissociation of subunit VIIa are coupled events that probably are responsible for the inactivation of cytochrome c oxidase. When cytochrome c oxidase is exposed to 500 microM hydrogen peroxide for 30 min at pH 7.4 and room temperature, subunits IV (modified up to 20%) and VIIc (modified up to 70%) each have an increased mass of 16 Da as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization mass spectrometry. In each case, the increased mass is caused by oxidation of a tryptophan (Trp19 within subunit VIIc and Trp48 within subunit IV), almost certainly due to formation of hydroxytryptophan. We conclude that hydrogen peroxide-induced oxidation of tryptophan and cardiolipin proceeds via the binuclear center since both modifications are prevented if the binuclear center is first blocked with cyanide. Bound cardiolipin and oxidized tryptophans are localized relatively far from the binuclear center (30-60 A); therefore, oxidation probably occurs by migration of a free radical generated at the binuclear center to these distal reaction sites.     

Tissue-specificity overrides species-specificity in cytoplasmic cytochrome c oxidase polypeptides

With a high-resolving dodecyl sulfate electrophoretic system rat liver cytochrome c oxidase was separated into 13 different polypeptides. An antiserum against rat liver holocytochrome c oxidase immunoreacted with all 13 polypeptides, as demonstrated by immunofluorescence after transfer of the separated Coomassie blue-stained bands on nitrocellulose and coupling with FITC-protein A ("western blot"). Polypeptide-specific antisera reacted only with their corresponding polypeptides indicating that the various protein bands are represented by individual polypeptides. From total proteins of rat liver, kidney, heart, spleen and skeletal muscle mitochondria, only the cytochrome c oxidase polypeptides showed immunofluorescence with an antiserum against the rat liver holoenzyme. In contrast to the polypeptide from liver, polypeptide VIa from heart and skeletal muscle showed little or no reactivity, indicating a tissue-specificity of this polypeptide. Mitochondrial proteins from pig, bovine and blackbird heart were incubated with an antiserum against the rat liver holoenzyme. Immunoreaction was found with most cytochrome c oxidase polypeptides but not with polypeptide VIa. This result demonstrates less immunological relationship between tissue-specific polypeptides (VIa, VIIa and VIII) of the same species than between tissue-unspecific polypeptides of different species.     

Orientation of rat liver cytochrome c oxidase subunits investigated with subunit-specific antisera

The orientation of rat liver cytochrome c oxidase subunits in the inner mitochondrial membrane was investigated with monospecific antisera against subunit II and nine nuclear-coded subunits. Mitoplasts were incubated with the antisera and the amount of bound antibodies was determined either directly with fluorescein-conjugated protein A or indirectly by back-titration of unbound antibodies with a nitrocellulose immunoassay. All subunits were found oriented to the cytosolic side, except subunits VIb and VIIc which did not react with their corresponding antisera. Antisera against subunits I, III and Vb were not available.     

The nuclear-coded subunits of yeast cytochrome c oxidase. The amino acid sequences of subunits VII and VIIa, structural similarities between the three smallest polypeptides of the holoenzyme, and implications for biogenesis

The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis.     

Contribution of peroxidized cardiolipin to inactivation of bovine heart cytochrome c oxidase

The lipid-soluble peroxides, tert-butyl hydroperoxide and peroxidized cardiolipin, each react with bovine cytochrome c oxidase and cause a loss of electron-transport activity. Coinciding with loss of activity is oxidation of Trp19 and Trp48 within subunits VIIc and IV, and partial dissociation of subunits VIa and VIIa. tert-Butyl hydroperoxide initiates these structural and functional changes of cytochrome c oxidase by three mechanisms: (1) radical generation at the binuclear center; (2) direct oxidation of Trp19 and Trp48; and (3) peroxidation of bound cardiolipin. All three mechanisms contribute to inactivation since blocking a single mechanism only partially prevents oxidative damage. The first mechanism is similar to that described for hydrogen peroxide [Biochemistry43:1003-1009; 2004], while the second and third mechanism are unique to organic hydroperoxides. Peroxidized cardiolipin inactivates cytochrome c oxidase in the absence of tert-butyl hydroperoxide and oxidizes the same tryptophans within the nuclear-encoded subunits. Peroxidized cardiolipin also inactivates cardiolipin-free cytochrome c oxidase rather than restoring full activity. Cardiolipin-free cytochrome c oxidase, although it does not contain cardiolipin, is still inactivated by tert-butyl hydroperoxide, indicating that the other oxidation products contribute to the inactivation of cytochrome c oxidase. We conclude that both peroxidized cardiolipin and tert-butyl hydroperoxide react with and triggers a cascade of structural alterations within cytochrome c oxidase. The summation of these events leads to cytochrome c oxidase inactivation.     

Immunohistochemical analysis of muscle cytochromec oxidase deficiency in children

Despite the demonstration of a clear biochemical defect, the genetic alterations causing childhood forms of cytochromec oxidase (COX) deficiency remain unknown. The double genetic origin (nuclear and mitochondrial DNA), and the complexity of COX enzyme structure and regulation, indicate the need for genetic iinvestigations of the molecular structure of individual COX subunits. In the present study a new monoclonal antibody, which reacts exclusively with heart-type human COX subunit VIIa (VIIa-H), and other monoclonal antibodies against human COX subunits, were used in the immunohistochemical analysis of skeletal muscle from children with different forms of mitochondrial myopathy with COX deficiency. By immunohistochemical investigation a normal reaction was seenn with antibodies to COX subunits IV, Va+Vb, and VIa+VIc in all four cases, and in two cases with antibodies to COX VIIa-H and VIIa+VIIb. In muscle from a fatal infantile case with cardiac and skeletal muscle involvement, no immunohistochemical reaction was seen with the monoclonal antibody against the tissue-specific subunit VIIa-H. In muscle from an 11-year-old boy with exclusive muscular symptoms and signs, immunohistological reactions were absent with COX subunit VIIa-H and COX subunits VIIa+VIIb, and slightly decreased with COX subunit II, thus demonstrating a different molecular mechanism in each case. It is concluded that the molecular basis of COX deficiency in childhood may vary greatly between patients.     

A role for Pet100p in the assembly of yeast cytochrome c oxidase: interaction with a subassembly that accumulates in a pet100 mutant

The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins. One of these, Pet100p, is required for cytochrome c oxidase. Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis. Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane. We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII. Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme. In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI. Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits.     

Identification of bovine heart cytochrome c oxidase subunits modified by the lipid peroxidation product 4-hydroxy-2-nonenal

Bovine heart cytochrome c oxidase (CcO) was inactivated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in a time- and concentration-dependent manner with pseudo-first-order kinetics. Cytochrome c oxidase electron transport activity decreased by as much as 50% when the enzyme was incubated for 2 h at room temperature with excess HNE (300-500 microM). HNE-modified CcO subunits were identified by two mass spectrometric methods: electrospray ionization mass spectrometry (ESI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). All of the experimentally determined molecular masses were in excellent agreement with published sequence values with an accuracy of approximately 1 part per 10000 mass units for subunits smaller than 20 kDa and approximately 1 part per 1000 mass units for the three subunits larger than 20 kDa. Both MS methods detected six CcO subunits with an increased mass of 156 Da after reaction with HNE (subunits II, IV, Vb, VIIa, VIIc, and VIII); this result indicates a single Michael-type reaction site on either a lysine or histidine residue within each subunit. Reaction of HNE with either subunit VIIc or subunit VIII (modified approximately 30% and 50-75%, respectively) must be responsible for CcO inhibition. None of the other subunits were modified more than 5% and could not account for the observed loss of activity. Reaction of HNE with His-36 of subunit VIII is most consistent with the approximately 50% inhibition of CcO: (1) subunit VIII is modified more than any other subunit by HNE; (2) the time dependence of subunit VIII modification is consistent with the percent inhibition of CcO; (3) His-36 was identified as the HNE-modified amino acid residue within subunit VIII by tandem MS analysis.     

Photolabeling of cardiolipin binding subunits within bovine heart cytochrome c oxidase

Subunits located near the cardiolipin binding sites of bovine heart cytochrome c oxidase (CcO) were identified by photolabeling with arylazido-cardiolipin analogues and detecting labeled subunits by reversed-phase HPLC and HPLC-electrospray ionization mass spectrometry. Two arylazido-containing cardiolipin analogues were synthesized: (1) 2-SAND-gly-CL with a nitrophenylazido group attached to the polar headgroup of cardiolipin (CL) via a linker containing a cleavable disulfide; (2) 2',2'-bis-(AzC12)-CL with two of the four fatty acid tails of cardiolipin replaced by 12-(N-4-azido-2-nitrophenyl) aminododecanoic acid. Both arylazido-CL derivatives were used to map the cardiolipin binding sites within two types of detergent-solubilized CcO: (1) intact 13-subunit CL-containing CcO (three to four molecules of endogenous CL remain bound per CcO monomer); (2) 11-subunit CL-free CcO (subunits VIa and VIb are missing because they dissociate during CL removal). Upon the basis of these photolabeling studies, we conclude that (1) subunits VIIa, VIIc, and possibly VIII are located near the two high-affinity cardiolipin binding sites, which are present in either form of CcO, and (2) subunit VIa is located adjacent to the lower affinity cardiolipin binding site, which is only present in the 13-subunit form of CcO. These data are consistent with the recent CcO crystal structure in which one cardiolipin is located near subunit VIIa and a second is located near subunit VIa (PDB ID code referenced in Tomitake, T. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 15304-15309). However, we propose that a third cardiolipin is bound between subunits VIIa and VIIc near the entrance to the D-channel. Cardiolipin bound at this location could potentially function as a proton antenna to facilitate proton entry into the D-channel. If true, it would explain the CcO requirement of bound cardiolipin for full electron transport activity.     

The role of subunit III in bovine cytochrome c oxidase. Comparison between native, subunit III-depleted and Paracoccus denitrificans enzymes

In order to obtain information on the role of subunit III in the function and aggregation state of cytochrome c oxidase, the kinetics of ferrocytochrome c oxidation by the bovine cytochrome c oxidase depleted of its subunit III were studied and compared with those of the oxidase isolated from P. denitrificans which contains only two subunits. The aggregation state of both enzymes dispersed in dodecyl maltoside was also compared. The two-subunit oxidase from P. denitrificans gave linear Eadie-Hofstee plots and the enzyme resulted to be monomeric (Mr = 82 000) both, in gel filtration and sucrose gradient centrifugation studies. The bovine heart subunit III depleted enzyme, under conditions when the P. denitrificans cytochrome c oxidase was in the form of monomers, was found to be dimeric by sucrose gradient centrifugation analysis. At lower enzyme concentrations monomers were, however, detected by gel filtration. Depletion of subunit III was accompanied by the loss of small polypeptides (VIa, VIb and VIIa) and of almost all phospholipid (1-2 molecules were left per molecule of enzyme). The electron-transfer activity of the subunit III-depleted enzyme showed a monophasic Eadie-Hofstee plot, which upon addition of phospholipids became non-linear, similar to that of the control bovine cytochrome c oxidase. One of the roles of subunit III may be that of stabilising the dimers of cytochrome c oxidase. Lack of this subunit and loss of phospholipid is accompanied by a change in the kinetics of electron transfer, which might be the consequence of enzyme monomerisation.     

Regulation by heme of the synthesis of cytochrome C oxidase subunits V and VII in yeast

The effects of heme on the synthesis of subunits V and VII of cytochrome c oxidase were examined in the heme-deficient yeast mutant, GL1. In vitro translation and immunodetection with subunit-specific antisera indicated a 50% decrease in both proteins, with RNA obtained from hemeless cells. Unsupplemented, pulse-labeled cells contained both V and VII polypeptides, but at extremely low levels as compared with those found in delta-aminolevulinic acid-supplemented cells. The data suggest that heme controls the formation of mRNAs for the two subunits, and may also have a regulatory role in translation and in the stability of the polypeptides.     

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.     

Expression of cloned mitochondrial DNA from the petite negative yeast Schizosaccharomyces pombe in E. coli minicells

The minicell producing Escherichia coli strain D24 (lysogenic for phage lambda cI857) was transformed with the recombinant plasmid pDG3 containing the entire mitochondrial (mt) genome of the fission yeast Schizosaccharomyces pombe (S. pombe) cloned in the single BamHI-site of the E. coli plasmid pBR322 (Del Giudice 1981). By DNA-RNA hybridization it could be shown that the total mtDNA sequence of the plasmid pDG3 was transcribed in the E. coli minicells. The cloned mtDNA also directed the synthesis of at least five novel polypeptides with molecular weights between 7,200 and 34,000. When the minicell producing E. coli strain P678-54 was transformed with the hybrid plasmid pDG3, considerable portions of the inserted mtDNA sequences were deleted. One of the resulting plasmids (pDG4), lacking about two-thirds of the mtDNA sequence, directed the synthesis of new polypeptides in the range of 7,000 to 17,500 daltons. Another derivative of pDG3, the plasmid pDG5, containing one-sixth of the mtDNA sequence, directed the synthesis of at least three novel polypeptides. The mt origin of novel polypeptides coded by the hybrid plasmid pDG3 was demonstrated by use of antisera raised against total mitochondrial proteins from S. pombe and antisera against subunits II and III of cytochrome c oxidase from Saccharomyces cerevisiae (S. cer.).     

Vertebrate myosin VIIb is a high duty ratio motor adapted for generating and maintaining tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.