Methods for Assessing Mitochondrial Function in Diabetes

A growing body of research is investigating the potential contribution of mitochondrial function to the etiology of type 2 diabetes. Numerous in vitro, in situ, and in vivo methodologies are available to examine various aspects of mitochondrial function, each requiring an understanding of their principles, advantages, and limitations. This review provides investigators with a critical overview of the strengths, limitations and critical experimental parameters to consider when selecting and conducting studies on mitochondrial function. In vitro (isolated mitochondria) and in situ (permeabilized cells/tissue) approaches provide direct access to the mitochondria, allowing for study of mitochondrial bioenergetics and redox function under defined substrate conditions. Several experimental parameters must be tightly controlled, including assay media, temperature, oxygen concentration, and in the case of permeabilized skeletal muscle, the contractile state of the fibers. Recently developed technology now offers the opportunity to measure oxygen consumption in intact cultured cells. Magnetic resonance spectroscopy provides the most direct way of assessing mitochondrial function in vivo with interpretations based on specific modeling approaches. The continuing rapid evolution of these technologies offers new and exciting opportunities for deciphering the potential role of mitochondrial function in the etiology and treatment of diabetes

Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome c oxidase

Redox-driven proton pumps such as cytochrome c oxidase (CcO) are fundamental elements of the energy transduction machinery in biological systems. CcO is an integral membrane protein that acts as the terminal electron acceptor in respiratory chains of aerobic organisms, catalyzing the four-electron reduction of O(2) to H(2)O. This reduction also requires four protons taken from the cytosolic or negative side of the membrane, with an additional uptake of four protons that are pumped across the membrane. Therefore, the proton pump must embody a “gate,” which provides alternating access of protons to one or the other side of the membrane but never both sides simultaneously. However, the exact mechanism of proton translocation through CcO remains unknown at the molecular level. Understanding pump function requires knowledge of the nature and location of these structural changes that is often difficult to access with crystallography or NMR spectroscopy. In this paper, we demonstrate, with amide hydrogen/deuterium exchange MS, that transitions between catalytic intermediates in CcO are orchestrated with opening and closing of specific proton pathways, providing an alternating access for protons to the two sides of the membrane. An analysis of these results in the framework of the 3D structure of CcO indicate the spatial location of a gate, which controls the unidirectional proton flux through the enzyme and points to a mechanism by which CcO energetically couples electron transfer to proton translocation

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O(2). The reduction of O(2), accompanied by the uptake of substrate protons to form H(2)O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H(2)O the uptake and release reactions occur with the same rates, in D(2)O the substrate and pumped protons are taken up first (τ(D) ≅ 200 μs, “peroxy” to “ferryl” transition) followed by a significantly slower proton release to the other side of the membrane (τ(D) ≅ 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation

Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560–3,800 cm-1 range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(−), and 3,607(+) cm-1. A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D2O and H218O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O(2)-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located ≈30 Å from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK(a) of an internal proton donor, Glu-286, from ≈9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N/N139D double-mutant CcO) results in restoration of the Glu-286 pK(a) to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N/N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P → F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process

A mitochondrial DNA mutation linked to colon cancer results in proton leaks in cytochrome c oxidase

An increasing number of cancer types have been found to be linked to specific mutations in the mitochondrial DNA, which result in specific structural changes of the respiratory enzyme complexes. In this study, we have investigated the effect of 2 such mutations identified in colon cancer patients, leading to the amino acid substitutions Ser458Pro and Gly125Asp in subunit I of cytochrome c oxidase (complex IV) [Greaves et al. (2006) Proc Natl Acad Sci USA 103:714–719]. We introduced these mutations in Rhodobacter sphaeroides, which carries an oxidase that serves as a model of the mitochondrial counterpart. The lack of expression of the former variant indicates that the amino acid substitution results in severely altered overall structure of the enzyme. The latter mutation (Gly171Asp in the bacterial oxidase) resulted in a structurally intact enzyme, but with reduced activity (approximately 30%), mainly due to slowed reduction of the redox site heme a. Furthermore, even though the Gly171Asp CytcO pumps protons, an intrinsic proton leak was identified, which would lead to a decreased overall energy-conversion efficiency of the respiratory chain, and would also perturb transport processes such as protein, ion, and metabolite trafficking. Furthermore, the specific leak may act to alter the balance between the electrical and chemical components of the proton electrochemical gradient

Ion exclusion by sub-2-nm carbon nanotube pores

Biological pores regulate the cellular traffic of a large variety of solutes, often with high selectivity and fast flow rates. These pores share several common structural features: the inner surface of the pore is frequently lined with hydrophobic residues, and the selectivity filter regions often contain charged functional groups. Hydrophobic, narrow-diameter carbon nanotubes can provide a simplified model of membrane channels by reproducing these critical features in a simpler and more robust platform. Previous studies demonstrated that carbon nanotube pores can support a water flux comparable to natural aquaporin channels. Here, we investigate ion transport through these pores using a sub-2-nm, aligned carbon nanotube membrane nanofluidic platform. To mimic the charged groups at the selectivity region, we introduce negatively charged groups at the opening of the carbon nanotubes by plasma treatment. Pressure-driven filtration experiments, coupled with capillary electrophoresis analysis of the permeate and feed, are used to quantify ion exclusion in these membranes as a function of solution ionic strength, pH, and ion valence. We show that carbon nanotube membranes exhibit significant ion exclusion that can be as high as 98% under certain conditions. Our results strongly support a Donnan-type rejection mechanism, dominated by electrostatic interactions between fixed membrane charges and mobile ions, whereas steric and hydrodynamic effects appear to be less important