Complex I Mutant Gas-1 (fc21)
In the absence of isoflurane, complexes I and I–III were severely depressed by the gas-1 mutation alone: 62 nmol/min/mg and 85 nmol/min/mg, respectively, which are 25% and 8% of the wildtype level (Fig. 2, A and B, Table 1). Isoflurane minimally inhibited these 2 assays in gas-1.
NFR activity in the mutant was about 60% of wildtype (Fig. 2F). As in wildtype, NFR activity in gas-1 was not inhibited by isoflurane. Hence, the presence of a mutant subunit in the neighboring iron–sulfur–protein subcomplex does not induce a new target for isoflurane on the flavoprotein. Complexes II–III activity of gas-1 was similar to that of wildtype (Fig. 2C), as can be expected for an activity that does not involve the mutated peptide. Complexes III and IV were not studied in gas-1 because the mutation does not change these activities from wildtype, and the wildtype activities are not inhibited by isoflurane.
We have shown for the first time that isoflurane inhibition of complex I, the most susceptible member of the ETC to inhibition by VAs, is not due to competition of the agent for the hydrophobic binding site of UQ within complex I. In addition, it appears that isoflurane has no effect on the flow of electrons through the flavoprotein, the proximal tip of the matrix arm of complex I that protrudes into the aqueous matrix of the mitochondrion. Enzymatic rates of both complex I and complexes I–III were very sensitive to inhibition by isoflurane. Also, complex I activity is measured using a water-soluble form of UQ, whereas I–III activity uses the native quinone. Because both I and I–III activity are inhibited by isoflurane, anesthetic inhibition of complex I does not involve the binding sites for the UQ isoprenyl tail.a
Our results are fully consistent with selective inhibition of complex I by isoflurane at a site distal to the flavoprotein subcomplex (Fig. 1E). Considering that both cytochrome c reductase assays (I–III Fig. 2B, II–III Fig. 2C) use a shared electron path from UQ to cytochrome c, the marked difference in sensitivity to isoflurane confirms that complex I is more sensitive to the drug than are complexes II and III. Our results for isoflurane inhibition of complex I in C. elegans are in agreement with previous findings for halothane in rat liver mitochondria30 and for 3 VAs, including isoflurane, in pig heart mitochondria.31 However, these earlier reports did not establish IC50s for isoflurane, nor did they definitively establish that the complex I inhibition was selective to complex I or a portion of complex I. Our study, because it interrogates each individual relevant step of electron flow, represents a comprehensive evaluation of the possible indirect effects of isoflurane on complex I that have not been previously reported.
Likewise, previous investigations of the affects of VAs on ETCs preceded our current knowledge of the importance of supercomplex I:III:IVn for proper complex I function. In vivo complexes I, III, and IV form a supercomplex that is thought to be the functional unit of respiration.17–19 Normal associations with complex IV and complex III are necessary for maximal electron transport activity of fully assembled complex I in vitro.17–20 The data indicate that supercomplexes remain intact in ETC assays and that complexes III and IV can indirectly affect complex I activity. Specific interactions of VAs with complexes III and IV have been convincingly demonstrated by Xi et al. using radiolabeled halothane.21 It is therefore also conceivable that anesthetic bound to complex III or IV could elicit activity changes in complex I (Fig. 1B). However, such an interaction would be expected to also affect the activity of complex III or IV. Our results indicate that neither complex III nor complex IV is the relevant target for isoflurane. Alternatively, isoflurane might bind to complexes III and IV without affecting their individual activities but indirectly inhibit complex I by destabilizing activating interactions between complex I and its partners (Fig. 1C). Weakened interactions between members of the supercomplexes that are not detected by 1-dimensional BNGs can be unmasked by 2-dimensional (2D) BNGs. We have identified mutations in subunits of complex III that cause an ∼40% decrease in complex I activity and a striking vulnerability of the I:III:IV supercomplex to dissociation by maltoside,26 conditions identical to those we used for our 2D native gels presented here. If isoflurane inhibited complex I by weakening the association of complexes within the supercomplex, we would expect an observable effect on stability after exposure to 19% isoflurane. However, 2D electrophoresis did not reveal any stabilizing or destabilizing effects of 19% isoflurane on the supercomplexes. Thus, isoflurane did not disrupt the binding of complexes I, III, and IV within the supercomplex. These results further validate the conclusion drawn from the ETC assays, that isoflurane interacts directly with complex I.
VAs are a heterogeneous group of chemicals, not identified by a common active chemical group. Their anesthetic potency is largely determined by their gas/oil partitioning coefficient.32 The binding sites for UQ and 3 classes of complex I inhibitors are believed to be located in a hydrophobic fold of the complex.12,33,34 UQ ordinarily has a long hydrophobic polyisoprenyl tail thought to be embedded in the mitochondrial membrane, as well as a relatively hydrophilic head portion that swings between hydrophilic domains of complex I and complex III, passing electrons through the ETC.12,33,34 In the prokaryote Thermus thermophilus, crystallographic data demonstrate that the binding site for quinone in complex I is sufficiently large to chamber VA molecules.9,35 In addition, that part of the protein Nqo4 (also termed 49 kDa), an orthologue of GAS-1, which binds to UQ, is a 4 α-helix bundle, located precisely at the junction of the transmembrane and the hydrophilic domains that compose the quinone binding site. This type of structure, a 4 α-helix bundle, has been used extensively to model the binding of VAs to membrane proteins.15,16 Thus, the hydrophobic binding pocket, containing the UQ binding site, could be a prime candidate for the binding of VAs. Just as some of the inhibitor binding sites of complex I overlap with each other and with UQ binding sites, it is easily conceivable that anesthetics interfere with the binding of UQ, and vice versa. However, such competitive inhibition is unequivocally excluded (Fig. 4, Table 2) because even very high concentrations of decylubiquinone could not out-compete a fixed concentration (5.7%) of isoflurane. We conclude that isoflurane and UQ neither share a common binding site nor bind to overlapping sites (Fig. 1, D and E). Our results furthermore excluded another variant of competitive inhibition: heterotropic allosteric inhibition. Binding of either isoflurane or UQ does not change the conformation of the complex as to negatively affect binding of the other. However, it is likely that isoflurane binds to other sites within complex I, the I:III:IV supercomplex, or even to UQ itself.
Once kinetics indicated that cooperative binding of UQ had to be considered, the simple model to distinguish the remaining 2 mechanisms of inhibition was no longer applicable. Thus, we were unable to determine whether isoflurane binds and blocks exclusively a preformed enzyme-substrate complex (uncompetitive inhibition) or binds to complex I independent of UQ (noncompetitive inhibition). Having established wildtype complex I as a selective target for isoflurane, but not in direct competition with Q for a binding site, we tested whether the hypersensitivity of gas-1 may be caused directly by hypersensitivity of its mutant complex I to isoflurane.
GAS-1, a core subunit of complex I, is 83.4% similar, at the amino acid level, to its human counterpart.13,14 The fc21 missense mutation (R290K) affects a residue in GAS-1 that is conserved even in simple bacterial NADH:UQ reductases. It renders C. elegans exquisitely sensitive to all Vas, but especially so to isoflurane (wildtype EC50 = 7.0 ± 0.1%, fc21 EC50 = 1.3 ± 0.2%).1,10 At 7.0% isoflurane, wildtype complex I is severely inhibited to about 40% of its potential activity, suggesting that the inhibition may cause immobility of the animal. However, this argument loses traction when results from the mutant are considered. The baseline activity of mutant complex I was very low, yet gas-1 is quite mobile in the absence of isoflurane. Its complex I activity measured in the absence of anesthetic is already lower than that of the wildtype mitochondria in the presence of anesthetizing concentrations of isoflurane. A similar pattern as described here for isoflurane has previously been found for halothane.36 Thus, there is no common threshold of complex I activity (measured in vitro) for wildtype and mutant, below which the worms become immobile/anesthetized.
It would appear that although complex I baseline activity (before inhibition) is a fair predictor of the anesthetic sensitivity of an organism,4 the absolute level of complex I activity (resulting from the combination of genetic background and inhibition by anesthetic) is a poor predictor of the anesthetic state of the animal. However, several points indicate that complex I is important in determining anesthetic sensitivity. First, when complex I activity is inhibited by knockdown of multiple subunits of complex I, anesthetic sensitivity is almost uniformly increased.14 Second, children with complex I defects have been noted to have an increased sensitivity to VAs.5 Third, as shown here and by others, of all complexes in the ETC, complex I is by far the most sensitive to VAs.30,31,37 Finally, as shown in this study, complex I activity in gas-1 was resistant to further depression by the anesthetic (the isoflurane IC50 for gas-1 > that for N2). Thus, although isoflurane does not compete directly with quinone for binding to complex I, a mutation in a protein that is part of the UQ binding site does cause resistance to inhibition. The simplest interpretation of these data is that the mutation alters an isoflurane target.
How can one reconcile that complex I dysfunction leads to anesthetic hypersensitivity, yet we cannot correlate levels of complex I activity with the anesthetic state? First, defects in complex I, which changes its sensitivity to isoflurane, may lead to more important secondary changes in pertinent downstream anesthetic targets, resulting in an increase in anesthetic sensitivity. Second, ETC assays measure maximal enzymatic activity, a condition probably not attained in vivo. N2 and gas-1 animals could have similar basal rates of complex I function in vivo. However, this rate may approach gas-1's maximal complex I function and render the mutant complex I more vulnerable than that of wildtype to even small decreases in capacity. This difference could result, in the presence of isoflurane, in a more rapid decrease by gas-1 than N2 to a common in vivo complex I threshold for immobility. Third, chronic loss of complex I activity (as in gas-1) may lead to compensatory changes that allow the animal to function at low complex I rates not tolerated by the wildtype animal during acute anesthetic exposure. For example, the capacity for complex II–dependent respiration is up in gas-110, and we have shown here that complex II activity is not inhibited by isoflurane. Fourth, the anesthetic state may be the result of inhibition of multiple types of targets, of which mitochondria are only one. Studies to differentiate between these possibilities are underway.
Finally, we have found that UQ itself has a cooperative interaction with complex I not previously appreciated. To our knowledge we are the first to report non-Michaelis–Menten behavior of complex I. For complex I the existence of multiple UQ binding sites is supported by experimental evidence and demanded by several mechanistic models;38,39 however, their exact number is still a matter of debate.40 Tocilescu et al. identified residues on the 49 kDa subunit (the GAS-1 orthologue) and the adjacent subunit (PSST) that altered the interaction of complex I with UQ.12 However, this work did not establish whether more than 1 UQ molecule may occupy this redox active site, nor has it been excluded that additional UQ binding sites may exist elsewhere on complex I, which may be merely allosteric. Our measured Hill coefficients of 2.1 ± 0.2 and 2.5 ± 0.4 are consistent with either 2 strongly cooperative binding sites for UQ or more than 2, but weaker, interacting sites. It is noteworthy that isoflurane does not interrupt the cooperative interaction between UQ molecules because the Hill coefficient remains unchanged by the presence of isoflurane. It is crucial to acknowledge, however, that UQ kinetics are not trivial and are affected by multiple complicated interactions.41 The apparent cooperation we observe may be specific to C. elegans or even an artifact of our system. The water-soluble substrate decylubiquinone, which is used for this assay, may have unique properties of binding that are different from the native quinone. Such studies should be repeated in a well-defined system such as bovine mitochondrial preparations to clarify these possibilities.
In summary, we have shown that, in nematodes, isoflurane directly inhibits complex I function, at a site distal to the flavoprotein within the complex. We have excluded our initial hypothesis that the VAs compete with quinones for a hydrophobic binding site on complex I. The demonstrated stability of supercomplexes in the presence of isoflurane supported that complex I inhibition is not caused by isoflurane disrupting necessary interaction with neighboring complexes III and IV. Although complex I activity does predict VA sensitivity, absolute rates of complex I activity do not predict a threshold that correlates with immobility of the animal. Furthermore, we describe non-Michaelis–Menten kinetics, suggesting interacting binding sites, for UQ within complex I.
Name: Ernst-Bernhard Kayser, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Ernst-Bernhard Kayser has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Wichit Suthammarak, PhD.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Wichit Suthammarak has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
Name: Phil G. Morgan, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Phil G. Morgan has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Margaret M. Sedensky, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Margaret M. Sedensky has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
“Modified Hill Equation” to describe sigmoidal kinetics:
Where velocity v denotes the initial rate of product formation (here: NAD+) when the substrate (here: decylubiquinone) is present at a concentration of [S]. Parameters Vlim, K, h are constants: Vlim is the limiting velocity if enzyme could be fully saturated with substrate; K is the substrate concentration at which halfmaximal velocity is achieved, and h is the so-called Hill coefficient. Note that for the special case of h=1, the equation simplifies to describe hyperbolic (Michaelis–Menten) kinetics.
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© 2011 International Anesthesia Research Society
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