Browsing by Subject "Bioenergetics"

Electron bifurcation oxidizes a two-electron donor, using the two electrons to reduce high- and low-potential acceptors. Thus, one electron may move thermodynamically uphill, being kinetically coupled to the downhill flow of the other electron. Electron bifurcation in nature is often reversible (∆G ≈ 0) so minimal free energy is dissipated, and the reaction occurs at minimal overpotential. Thus, electron bifurcation is a compelling target for bioinspired catalysis and/or nanoscale device design.We formulate a general theory of the electron bifurcation process, using a many-electron hopping kinetics model with hopping rate constants estimated with thermally activated electron tunneling theory. We conclude that efficient and reversible electron bifurcation requires only a conserved redox potential (free energy) landscape, with steep redox potential gradients in the high- and low-potential branches (the reversible EB scheme). This energy landscape naturally builds up electron and hole populations near the bifurcating two-electron cofactor in the high- and low-potential branches, respectively, thus disfavoring short-circuiting electron-hole combination. The reversible EB scheme suppresses short-circuiting reactions by erecting a Boltzmann penalty against redox states of the enzyme that may short-circuit, and largely accounts for short-circuit insulation in complex III of the electron transport chain, although our model does not uniquely account for the slow short-circuit turnover with an inhibited low-potential branch. For electron bifurcating enzymes that reduce the low-potential substrate directly (such as bifurcating electron transfer flavoproteins), we hypothesize that a downward shift in redox potential of the low-potential substrate upon binding to the bifurcating enzyme (similar to the iron protein bound to nitrogenase) could explain how the requisite steep redox potential gradient is achieved without housing a series of redox cofactors in the low-potential branch. Electron bifurcating enzymes in nature are often found with bifurcating cofactors (for example quinones and flavins) with inverted reduction potentials (i.e., the first reduction potential lower than the second). We derive a free energy decomposition scheme for the half-reactions of a two electron species from quantum chemical calculations to find physical and chemical factors that determine whether the reduction potentials are inverted. Remarkably, two electron species such as quinones and flavins can exhibit normally-ordered or inverted reduction potentials depending on the protein environment. Using our energy decomposition scheme and an estimate of a quinone Pourbaix diagram under continuum mean-field environments with varying electrostatic permittivity, we conclude that the proton transfer events that often accompany reduction in addition to the electrostatic interactions of charged species may have a significant impact on the invertedness of two-electron compounds. Thus, we hypothesize that electrostatic interactions (including the self-interaction of a charged semiquinone) may principally explain the ability of flavins and quinones to change the order of their first and second reduction potentials so profoundly. Future studies will be required to test this hypothesis. In addition, we show that efficient and reversible electron bifurcation is possible with normally ordered potentials at the bifurcating cofactor, provided the absolute value of the difference between the first and second reduction potentials is large (on the order of the redox potential span of the high- and low-potential branches in the reversible EB scheme). This finding has implications for synthetic electron bifurcation, as engineering redox active catalytic sites with strongly normally ordered potentials seems more straightforward than sites with strongly inverted potentials. Finally, we describe kinetics schemes for thermodynamically irreversible electron bifurcation that rely on disequilibrium populations of electrons within the high potential branch (irreversible confurcation is possible with a disequilibrium population of holes within the low-potential branch). Although these schemes are yet only hypothesized, these schemes allow orders-of-magnitude regulation of the bifurcating turnover rate with the redox poise of the two-electron donor (including faster turnover than in the reversible EB scheme), with kinetics fit by a generalized Shockley ideal diode equation.

Heart failure is a worldwide public health problem with substantial clinical burden and economic costs. In the progression into failure, the heart undergoes dramatic alterations in mitochondrial fuel metabolism and bioenergetics. As such, there is considerable interest in the delineation of regulatory events involved in the metabolic dysfunction of heart failure. Previous collaborative work identified three metabolic signatures associated with early stage heart failure: 1) accumulation of acylcarnitine metabolites; 2) mitochondrial hyperacetylation; and 3) elevated ketone catabolism. The goal of this dissertation was to explore the role of these metabolic signatures in the pathogenesis of heart failure.

Tissue accumulation of acylcarnitine metabolites is characteristic of mitochondrial dysfunction and indicative of incomplete β-oxidation. This occurs when a large portion of the fatty acids (i.e., acyl groups) within the mitochondria are not fully catabolized and the resulting intermediates are transferred to carnitine esters, enabling the traversal of biological membranes and departure from the mitochondrial matrix.

Nϵ-acetylation in the mitochondrial matrix is a non-enzymatic, post-translational modification (PTM) that spontaneously arises from the relatively basic pH and abundance of acetyl-CoA. Accumulation of this PTM has been observed in other tissues and disease states with evidence suggesting it impairs mitochondrial metabolism and causes dysfunction. However, convincing studies are lacking to establish a direct causal connection between dysfunction and acetylation. To address this shortcoming, a novel assay platform for the comprehensive assessment of mitochondrial bioenergetic transduction was developed and validated. Next, we generated and validated a novel mouse model of cardiac mitochondrial hyperacetylation and utilized the bioenergetic assay platform to test the hypothesis that it causes metabolic perturbations. Surprisingly, these hyperacetylated mitochondria exhibited almost no deficits in mitochondrial oxidative metabolism. To determine if hyperacetylation causes mitochondrial dysfunction in vivo under pathologic stimuli, the mouse model and littermate controls were subjected to transaortic constriction, a surgical method to induce pressure-overload heart failure. The hyperacetylated animals did not exhibit enhanced sensitivity toward cardiac dysfunction relative controls. With these results, we concluded that mitochondrial hyperacetylation does not contribute to the pathogenesis of heart failure.

Elevated ketone catabolism was observed in early stage failing hearts. Through a series of murine and canine heart failure models, ketone catabolism was shown to be adaptive in response to pathological stress. Additionally, the mitochondrial bioenergetic assay platform was applied to cardiac mitochondria under substrate limited-conditions. These results indicate that ketone catabolism improves bioenergetic efficiency under constraints which mimic the failing heart. With these results, we conclude ketone catabolism is an important metabolic defense in response to the dysfunction of the failing heart.