Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease.

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of Paxlovid, a drug approved by the U.S. Food and Drug Administration for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here, we investigated the H172Y Mpro's conformational dynamics, folding stability, catalytic efficiency, and inhibitory activity using all-atom constant pH and fixed-charge molecular dynamics simulations, alchemical and empirical free energy calculations, artificial neural networks, and biochemical experiments. Our data suggest that the mutation significantly weakens the S1 pocket interactions with the N-terminus and perturbs the conformation of the oxyanion loop, leading to a decrease in the thermal stability and catalytic efficiency. Importantly, the perturbed S1 pocket dynamics weaken the nirmatrelvir binding in the P1 position, which explains the decreased inhibitory activity of nirmatrelvir. Our work demonstrates the predictive power of the combined simulation and artificial intelligence approaches, and together with biochemical experiments, they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the optimization of antiviral drugs. The presented approach, in general, can be applied to characterize mutation effects on any protein drug targets.

Structure-Kinetics Relationships of Opioids from Metadynamics and Machine Learning.

The nation's opioid overdose deaths reached an all-time high in 2021. The majority of deaths are due to synthetic opioids represented by fentanyl. Naloxone, which is an FDA-approved reversal agent, antagonizes opioids through competitive binding at the mu-opioid receptor (mOR). Thus, knowledge of opioid's residence time is important for assessing the effectiveness of naloxone. Here we estimated the residence times of 15 fentanyl and 4 morphine analogs using metadynamics, and compared them with the most recent measurement of the opioid kinetic, dissociation, and naloxone inhibitory constants (Mann, Li et al, Clin. Pharmacol. Therapeut. 2022). Importantly, the microscopic simulations offered a glimpse at the common binding mechanism and molecular determinants of dissociation kinetics for fentanyl analogs. The insights inspired us to develop a machine learning (ML) approach to analyze the kinetic impact of fentanyl's substituents based on the interactions with mOR residues. This proof-of-concept approach is general; for example, it may be used to tune ligand residence times in computer-aided drug discovery.

Structure-Kinetics Relationships of Opioids from Metadynamics and Machine Learning Analysis.

The nation's opioid overdose deaths reached an all-time high in 2021. The majority of deaths are due to synthetic opioids represented by fentanyl. Naloxone, which is a FDA-approved reversal agent, antagonizes opioids through competitive binding at the µ-opioid receptor (mOR). Thus, knowledge of the opioid's residence time is important for assessing the effectiveness of naloxone. Here, we estimated the residence times (τ) of 15 fentanyl and 4 morphine analogs using metadynamics and compared them with the most recent measurement of the opioid kinetic, dissociation, and naloxone inhibitory constants (Mann et al. Clin. Pharmacol. Therapeut. 2022, 120, 1020-1232). Importantly, the microscopic simulations offered a glimpse at the common binding mechanism and molecular determinants of dissociation kinetics for fentanyl analogs. The insights inspired us to develop a machine learning approach to analyze the kinetic impact of fentanyl's substituents based on the interactions with mOR residues. This proof-of-concept approach is general; for example, it may be used to tune ligand residence times in computer-aided drug discovery.

An Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease.

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds up in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here we investigated the H172Y Mpro's conformational dynamics, folding stability, catalytic efficiency, and inhibitory activity using all-atom constant pH and fixed-charge molecular dynamics simulations, alchemical and empirical free energy calculations, artificial neural networks, and biochemical experiments. Our data suggests that the mutation significantly weakens the S1 pocket interactions with the N-terminus and perturbs the conformation of the oxyanion loop, leading to a decrease in the thermal stability and catalytic efficiency. Importantly, the perturbed S1 pocket dynamics weakens the nirma-trelvir binding in the P1 position, which explains the decreased inhibitory activity of nirmatrelvir. Our work demonstrates the predictive power of the combined simulation and artificial intel-ligence approaches, and together with biochemical experiments they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the discovery of new antiviral drugs. The presented workflow can be applicable to characterize mutation effects on any protein drug targets.

How µ-opioid receptor recognizes fentanyl.

Roughly half of the drug overdose-related deaths in the United States are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, X-ray crystal structures of mOR in complex with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like opioids remains lacking. Exploiting the X-ray structure of BU72-bound mOR and several molecular simulation techniques, we elucidated the detailed binding mechanism of fentanyl. Surprisingly, in addition to the salt-bridge binding mode common to morphinan opiates, fentanyl can move deeper and form a stable hydrogen bond with the conserved His2976.52, which has been suggested to modulate mOR's ligand affinity and pH dependence by previous mutagenesis experiments. Intriguingly, this secondary binding mode is only accessible when His2976.52 adopts a neutral HID tautomer. Alternative binding modes may represent a general mechanism in G protein-coupled receptor-ligand recognition.

Kinetics and Mechanism of Fentanyl Dissociation from the µ-Opioid Receptor.

Driven by illicit fentanyl, opioid related deaths have reached the highest level in 2020. Currently, an opioid overdose is resuscitated by the use of naloxone, which competitively binds and antagonizes the µ-opioid receptor (mOR). Thus, knowledge of the residence times of opioids at mOR and the unbinding mechanisms is valuable for assessing the effectiveness of naloxone. In the present study, we calculate the fentanyl-mOR dissociation time and elucidate the mechanism by applying an enhanced sampling molecular dynamics (MD) technique. Two sets of metadynamics simulations with different initial structures were performed while accounting for the protonation state of the conserved H2976.52, which has been suggested to modulate the ligand-mOR affinity and binding mode. Surprisingly, with the Nδ-protonated H2976.52, fentanyl can descend as much as 10 Å below the level of the conserved D1473.32 before escaping the receptor and has a calculated residence time τ of 38 s. In contrast, with the Nϵ- and doubly protonated H2976.52, the calculated τ are 2.6 and 0.9 s, respectively. Analysis suggests that formation of the piperidine-Hid297 hydrogen bond strengthens the hydrophobic contacts with the transmembrane helix (TM) 6, allowing fentanyl to explore a deep pocket. Considering the experimental τ of ∼4 min for fentanyl and the role of TM6 in mOR activation, the deep insertion mechanism may be biologically relevant. The work paves the way for large-scale computational predictions of opioid dissociation rates to inform evaluation of strategies for opioid overdose reversal. The profound role of the histidine protonation state found here may shift the paradigm in computational studies of ligand-receptor kinetics.

How mu-Opioid Receptor Recognizes Fentanyl.

The opioid crisis has escalated during the COVID-19 pandemic. More than half of the overdose-related deaths are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, crystal structures of mOR complexed with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like synthetic opioids remains lacking. Exploiting the X-ray structure of mOR bound to a morphinan ligand and several state-of-the-art simulation techniques, including weighted ensemble and continuous constant pH molecular dynamics, we elucidated the detailed binding mechanism of fentanyl with mOR. Surprisingly, in addition to the orthosteric site common to morphinan opiates, fentanyl can move deeper and bind mOR through hydrogen bonding with a conserved histidine H297, which has been shown to modulate mOR's ligand affinity and pH dependence in mutagenesis experiments, but its precise role remains unclear. Intriguingly, the secondary binding mode is only accessible when H297 adopts a neutral HID tautomer. Alternative binding modes and involvement of tautomer states may represent general mechanisms in G protein-coupled receptor (GPCR)-ligand recognition. Our work provides a starting point for understanding mOR activation by fentanyl analogs that are emerging at a rapid pace and assisting the design of safer analgesics to combat the opioid crisis. Current protein simulation studies employ standard protonation and tautomer states; our work demonstrates the need to move beyond the practice to advance our understanding of protein-ligand recognition.

How µ-Opioid Receptor Recognizes Fentanyl.

In 2019, drug overdose has claimed over 70,000 lives in the United States. More than half of the deaths are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, the crystal structures of mOR in complex with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like synthetic opioids remains lacking. Exploiting the X-ray structure of mOR bound to a morphinan ligand and several state-of-the-art simulation techniques, including weighted ensemble and continuous constant pH molecular dynamics, we elucidated the detailed binding mechanism of fentanyl with mOR. Surprisingly, in addition to forming a salt-bridge with Asp1473.32 in the orthosteric site common to morphinan opiates, fentanyl can move deeper and bind mOR through hydrogen bonding with a conserved histidine His2976.52, which has been shown to modulate mOR's ligand affinity and pH dependence in mutagenesis experiments, but its precise role remains unclear. Intriguingly, the secondary binding mode is only accessible when His297 adopts a neutral HID tautomer. Alternative binding modes and involvement of tautomer states may represent general mechanisms in G protein-coupled receptor (GPCR)-ligand recognition. Our work provides a starting point for understanding the molecular basis of mOR activation by fentanyl which has many analogs emerging at a rapid pace. The knowledge may also inform the design of safer analgesics to combat the opioid crisis. Current protein simulation studies employ standard protonation and tautomer states; our work demonstrates the need to move beyond the practice to advance our understanding of protein-ligand recognition.

Aquaporin-7: A Dynamic Aquaglyceroporin With Greater Water and Glycerol Permeability Than Its Bacterial Homolog GlpF.

Xenopus oocytes expressing human aquaporin-7 (AQP7) exhibit greater osmotic water permeability and 3H-glycerol uptake vs. those expressing the bacterial glycerol facilitator GlpF. AQP7-expressing oocytes exposed to increasing extracellular [glycerol] under isosmolal conditions exhibit increasing swelling rates, whereas GlpF-expressing oocytes do not swell at all. To provide a structural basis for these observed physiological differences, we performed X-ray crystallographic structure determination of AQP7 and molecular-dynamics simulations on AQP7 and GlpF. The structure reveals AQP7 tetramers containing two monomers with 3 glycerols, and two monomers with 2 glycerols in the pore. In contrast to GlpF, no glycerol is bound at the AQP7 selectivity filter (SF), comprising residues F74, G222, Y223, and R229. The AQP7 SF is resolved in its closed state because F74 blocks the passage of small solutes. Molecular dynamics simulations demonstrate that F74 undergoes large and rapid conformational changes, allowing glycerol molecules to permeate without orientational restriction. The more rigid GlpF imposes orientational constraints on glycerol molecules passing through the SF. Moreover, GlpF-W48 (analogous to AQP7-F74) undergoes rare but long-lasting conformational changes that block the pore to H2O and glycerol.

Polyelectrolyte in Electric Field: Disparate Conformational Behavior along an Aminopolysaccharide Chain.

Electrical signals are increasingly used in fabrication of hydrogels (e.g., based on aminopolysaccharide chitosan) to guide the emergence of complex and anisotropic structure; however, how an imposed electric field affects the polymer chain conformation and orientation during the self-assembly process is not understood. Here, we applied nonequilibrium all-atom molecular dynamics simulations to explore the response of a charged chitosan chain comprising 5- or 20-monomer units to a constant uniform electric field in water and salt solution. While no conformational or orientational response was observed for the polyelectrolyte (PE) chains under the small electric fields within the simulation time, a field strength of 400 mV/nm induced significant changes. In water, a 5-mer chain is found to be slightly bent and oriented parallel to the field; however, surprisingly, a 20-mer chain displays candy-cane-like conformations whereby one half of the chain is collapsed and flexible, while the other half of the chain is stretched along the electric field. In salt solution, the disparity remains between the two halves of the 20-mer chain, although the backbone is extremely flexible with multiple bent regions and non-native conformations occur near the chain center in one of the three trajectories. The disparate conformational response along the polyelectrolyte chain may be attributed to the balancing forces between chain dynamics, electric polarization, counterion binding, and hydrodynamic pressure as well as friction. These findings reconcile existing experiments and theoretical studies and represent an important step toward understanding the complex roles of electric field and salt in controlling the structure and properties of soft matter.

The metabolic regulator Lamtor5 suppresses inflammatory signaling via regulating mTOR-mediated TLR4 degradation.

Comprehensive immune responses are essential for eliminating pathogens but must be tightly controlled to avoid sustained immune activation and potential tissue damage. The engagement of TLR4, a canonical pattern recognition receptor, has been proposed to trigger inflammatory responses with different magnitudes and durations depending on TLR4 cellular compartmentalization. In the present study, we identify an unexpected role of Lamtor5, a newly identified component of the amino acid-sensing machinery, in modulating TLR4 signaling and controlling inflammation. Specifically, Lamtor5 associated with TLR4 via their LZ/TIR domains and facilitated their colocalization at autolysosomes, preventing lysosomal tethering and the activation of mTORC1 upon LPS stimulation and thereby derepressing TFEB to promote autophagic degradation of TLR4. The loss of Lamtor5 was unable to trigger the TFEB-driven autolysosomal pathway and delay degradation of TLR4, leading to sustained inflammation and hence increased mortality among Lamtor5 haploinsufficient mice during endotoxic shock. Intriguingly, nutrient deprivation, particularly leucine deprivation, blunted inflammatory signaling and conferred protection to endotoxic mice. This effect, however, was largely abrogated upon Lamtor5 deletion. We thus propose a homeostatic function of Lamtor5 that couples pathogenic insults and nutrient availability to optimize the inflammatory response; this function may have implications for TLR4-associated inflammatory and metabolic disorders.

Characterization of Lipid-Protein Interactions and Lipid-Mediated Modulation of Membrane Protein Function through Molecular Simulation.

The cellular membrane constitutes one of the most fundamental compartments of a living cell, where key processes such as selective transport of material and exchange of information between the cell and its environment are mediated by proteins that are closely associated with the membrane. The heterogeneity of lipid composition of biological membranes and the effect of lipid molecules on the structure, dynamics, and function of membrane proteins are now widely recognized. Characterization of these functionally important lipid-protein interactions with experimental techniques is however still prohibitively challenging. Molecular dynamics (MD) simulations offer a powerful complementary approach with sufficient temporal and spatial resolutions to gain atomic-level structural information and energetics on lipid-protein interactions. In this review, we aim to provide a broad survey of MD simulations focusing on exploring lipid-protein interactions and characterizing lipid-modulated protein structure and dynamics that have been successful in providing novel insight into the mechanism of membrane protein function.

Selective Permeability of Carboxysome Shell Pores to Anionic Molecules.

Carboxysomes are closed polyhedral cellular microcompartments that increase the efficiency of carbon fixation in autotrophic bacteria. Carboxysome shells consist of small proteins that form hexameric units with semipermeable central pores containing binding sites for anions. This feature is thought to selectively allow access to RuBisCO enzymes inside the carboxysome by HCO3- (the dominant form of CO2 in the aqueous solution at pH 7.4) but not O2, which leads to a nonproductive reaction. To test this hypothesis, here we use molecular dynamics simulations to characterize the energetics and permeability of CO2, O2, and HCO3- through the central pores of two different shell proteins, namely, CsoS1A of α-carboxysome and CcmK4 of ß-carboxysome shells. We find that the central pores are in fact selectively permeable to anions such as HCO3-, as predicted by the model.

Microscopic view of lipids and their diverse biological functions.

Biological membranes and their diverse lipid constituents play key roles in a broad spectrum of cellular and physiological processes. Characterization of membrane-associated phenomena at a microscopic level is therefore essential to our fundamental understanding of such processes. Due to the semi-fluid and dynamic nature of lipid bilayers, and their complex compositions, detailed characterization of biological membranes at an atomic scale has been refractory to experimental approaches. Computational modeling and simulation offer a highly complementary toolset with sufficient spatial and temporal resolutions to fill this gap. Here, we review recent molecular dynamics studies focusing on the diversity of lipid composition of biological membranes, or aiming at the characterization of lipid-protein interaction, with the overall goal of dissecting how lipids impact biological roles of the cellular membranes.

Bacterial denitrifying nitric oxide reductases and aerobic respiratory terminal oxidases use similar delivery pathways for their molecular substrates.

The superfamily of heme­copper oxidoreductases (HCOs) include both NO and O2 reductases. Nitric oxide reductases (NORs) are bacterial membrane enzymes that catalyze an intermediate step of denitrification by reducing nitric oxide (NO) to nitrous oxide (N2O). They are structurally similar to heme­copper oxygen reductases (HCOs), which reduce O2 to water. The experimentally observed apparent bimolecular rate constant of NO delivery to the deeply buried catalytic site of NORs was previously reported to approach the diffusion-controlled limit (108-109 M-1 s-1). Using the crystal structure of cytochrome-c dependent NOR (cNOR) from Pseudomonas aeruginosa, we employed several protocols of molecular dynamics (MD) simulation, which include flooding simulations of NO molecules, implicit ligand sampling and umbrella sampling simulations, to elucidate how NO in solution accesses the catalytic site of this cNOR. The results show that NO partitions into the membrane, enters the enzyme from the lipid bilayer and diffuses to the catalytic site via a hydrophobic tunnel that is resolved in the crystal structures. This is similar to what has been found for O2 diffusion through the closely related O2 reductases. The apparent second order rate constant approximated using the simulation data is ~5 × 108 M-1 s-1, which is optimized by the dynamics of the amino acid side chains lining in the tunnel. It is concluded that both NO and O2 reductases utilize well defined hydrophobic tunnels to assure that substrate diffusion to the buried catalytic sites is not rate limiting under physiological conditions.

Cytochrome aa3 Oxygen Reductase Utilizes the Tunnel Observed in the Crystal Structures To Deliver O2 for Catalysis.

Cytochrome aa3 is the terminal respiratory enzyme of all eukaryotes and many bacteria and archaea, reducing O2 to water and harnessing the free energy from the reaction to generate the transmembrane electrochemical potential. The diffusion of O2 to the heme-copper catalytic site, which is buried deep inside the enzyme, is the initiation step of the reaction chemistry. Our previous molecular dynamics (MD) study with cytochrome ba3, a homologous enzyme of cytochrome aa3 in Thermus thermophilus, demonstrated that O2 diffuses from the lipid bilayer to its reduction site through a 25 Šlong tunnel inferred by Xe binding sites detected by X-ray crystallography [Mahinthichaichan, P., Gennis, R., and Tajkhorshid, E. (2016) Biochemistry 55, 1265-1278]. Although a similar tunnel is observed in cytochrome aa3, this putative pathway appears partially occluded between the entrances and the reduction site. Also, the experimentally determined second-order rate constant for O2 delivery in cytochrome aa3 (∼108 M-1 s-1) is 10 times slower than that in cytochrome ba3 (∼109 M-1 s-1). A question to be addressed is whether cytochrome aa3 utilizes this X-ray-inferred tunnel as the primary pathway for O2 delivery. Using complementary computational methods, including multiple independent flooding MD simulations and implicit ligand sampling calculations, we probe the O2 delivery pathways in cytochrome aa3 of Rhodobacter sphaeroides. All of the O2 molecules that arrived in the reduction site during the simulations were found to diffuse through the X-ray-observed tunnel, despite its apparent constriction, supporting its role as the main O2 delivery pathway in cytochrome aa3. The rate constant for O2 delivery in cytochrome aa3, approximated using the simulation results, is 10 times slower than in cytochrome ba3, in agreement with the experimentally determined rate constants.

Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase.

The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP+ in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across a narrow pore and a hydrophobic "dry" region in the middle of the membrane channel, with key residues His42α2 (chain A) being protonated and Thr214ß (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214ß to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH.

The cellular membrane as a mediator for small molecule interaction with membrane proteins.

The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

All the O2 Consumed by Thermus thermophilus Cytochrome ba3 Is Delivered to the Active Site through a Long, Open Hydrophobic Tunnel with Entrances within the Lipid Bilayer.

Cytochrome ba3 is a proton-pumping heme-copper oxygen reductase from the extreme thermophile Thermus thermophilus. Despite the fact that the enzyme's active site is buried deep within the protein, the apparent second order rate constant for the initial binding of O2 to the active-site heme has been experimentally found to be 10(9) M(-1) s(-1) at 298 K, at or near the diffusion limit, and 2 orders of magnitude faster than for O2 binding to myoglobin. To provide quantitative and microscopic descriptions of the O2 delivery pathway and mechanism in cytochrome ba3, extensive molecular dynamics simulations of the enzyme in its membrane-embedded form have been performed, including different protocols of explicit ligand sampling (flooding) simulations with O2, implicit ligand sampling analysis, and in silico mutagenesis. The results show that O2 diffuses to the active site exclusively via a Y-shaped hydrophobic tunnel with two 25-Å long membrane-accessible branches that coincide with the pathway previously suggested by the crystallographically identified xenon binding sites. The two entrances of the bifurcated tunnel of cytochrome ba3 are located within the lipid bilayer, where O2 is preferentially partitioned from the aqueous phase. The largest barrier to O2 migration within the tunnel is estimated to be only 1.5 kcal/mol, allowing O2 to reach the enzyme active site virtually impeded by one-dimensional diffusion once it reaches a tunnel entrance at the protein surface. Unlike other O2-utilizing proteins, the tunnel is "open" with no transient barriers observed due to protein dynamics. This unique low-barrier passage through the protein ensures that O2 transit through the protein is never rate-limiting.

Conformational coupling between the active site and residues within the K(C)-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase.

The respiratory chains of nearly all aerobic organisms are terminated by proton-pumping heme-copper oxygen reductases (HCOs). Previous studies have established that C-family HCOs contain a single channel for uptake from the bacterial cytoplasm of all chemical and pumped protons, and that the entrance of the K(C)-channel is a conserved glutamate in subunit III. However, the majority of the K(C)-channel is within subunit I, and the pathway from this conserved glutamate to subunit I is not evident. In the present study, molecular dynamics simulations were used to characterize a chain of water molecules leading from the cytoplasmic solution, passing the conserved glutamate in subunit III and extending into subunit I. Formation of the water chain, which controls the delivery of protons to the K(C)-channel, was found to depend on the conformation of Y241(Vc), located in subunit I at the interface with subunit III. Mutations of Y241(Vc) (to A/F/H/S) in the Vibrio cholerae cbb3 eliminate catalytic activity, but also cause perturbations that propagate over a 28-Å distance to the active site heme b3. The data suggest a linkage between residues lining the K(C)-channel and the active site of the enzyme, possibly mediated by transmembrane helix α7, which contains both Y241(Vc) and the active site cross-linked Y255(Vc), as well as two CuB histidine ligands. Other mutations of residues within or near helix α7 also perturb the active site, indicating that this helix is involved in modulation of the active site of the enzyme.