Effect of the mitochondrial membrane potential on the absorbance of the reduced form of cytochrome c oxidase.

The effect of mitochondrial membrane potential (ΔΨm) on the absorbance of the reduced cytochrome c oxidase (COX) was evaluated in isolated rabbit heart mitochondria using integrating sphere optical spectroscopy. Maximal reduction of the mitochondrial cytochromes was achieved by either blowing nitrogen to remove oxygen, or by adding cyanide. Gradual depolarization of ΔΨm by adding increasing concentrations of uncoupler resulted in an increase of up to 50 % in the absorbance of cytochrome aa3 under nitrogen saturation, and of 25 % with cyanide. Cytochrome aa3 absorbance increases were also observed in the presence of cyanide with apyrase (20 %) or oligomycin (12 %). The bL heme absorbance also decreased as expected from ΔΨm depolarization. A ~ 1 nm red shift in the peak wavelength of cytochrome aa3 was observed under anoxic conditions as ΔΨm was depolarized. Importantly, cytochrome c and c1 absorbances remained constant at levels corresponding to full reduction under all experimental manipulations of ΔΨm, especially with cyanide. These data suggest that ΔΨm-dependent changes in the absorbance of reduced COX were due to a variable extinction coefficient of heme a and/or a3 as a function of ΔΨm. A similar increase in the reduced cytochrome aa3 absorbance without changes in cytochrome c and c1 was observed in the perfused rabbit heart when decreasing ΔΨm with uncoupler. Our results imply that COX absorbance in its fully reduced state does not simply reflect the oxygen tension but also the ΔΨm. This may prove useful in monitoring ΔΨm under anoxic or ischemic conditions in intact tissue.

Lactate oxidation in Paracoccus denitrificans.

Paracoccus denitrificans has a classical cytochrome-dependent electron transport chain and two alternative oxidases. The classical transport chain is very similar to that in eukaryotic mitochondria. Thus, P. denitrificans can serve as a model of the mammalian mitochondrion that may be more tractable in elucidating mechanisms of regulation of energy production than are mitochondria. In a previous publication we reported detailed studies on respiration in P. denitrificans grown aerobically on glucose or malate. We noted that P. denitrificans has large stores of lactate under various growth conditions. This is surprising because P. denitrificans lacks an NAD+-dependent lactate dehydrogenase. The aim of this study was to investigate the mechanisms of lactate oxidation in P. denitrificans. We found that the bacterium grows well on either d-lactate or l-lactate. Growth on lactate supported a rate of maximum respiration that was equal to that of cells grown on glucose or malate. We report proteomic, metabolomic, and biochemical studies that establish that the metabolism of lactate by P. denitrificans is mediated by two non-NAD+-dependent lactate dehydrogenases. One prefers d-lactate over l-lactate (D-iLDH) and the other prefers l-lactate (L-iLDH). We cloned and produced the D-iLDH and characterized it. The Km for d-lactate was 34 µM, and for l-lactate it was 3.7 mM. Pyruvate was not a substrate, rendering the reaction unidirectional with lactate being converted to pyruvate for entry into the TCA cycle. The intracellular lactate was ∼14 mM such that both isomers could be metabolized by the enzyme. The enzyme has 1 FAD per molecule and utilizes a quinone rather than NAD + as an electron acceptor. D-iLDH provides a direct entry of lactate reducing equivalents into the cytochrome chain, potentially explaining the high respiratory capacity of P. denitrificans in the presence of lactate.

Cardiac nitric oxide scavenging: role of myoglobin and mitochondria.

Vascular production of nitric oxide (NO) regulates vascular tone. However, highly permeable NO entering the cardiomyocyte would profoundly impact metabolism and signalling without scavenging mechanisms. The purpose of this study was to establish mechanisms of cardiac NO scavenging. Quantitative optical studies of normoxic working hearts demonstrated that micromolar NO concentrations did not alter mitochondria redox state or respiration despite detecting NO oxidation of oxymyoglobin to metmyoglobin. These data are consistent with proposals that the myoglobin/myoglobin reductase (Mb/MbR) system is the major NO scavenging site. However, kinetic studies in intact hearts reveal a minor role (∼9%) for the Mb/MbR system in NO scavenging. In vitro, oxygenated mitochondria studies confirm that micromolar concentrations of NO bind cytochrome oxidase (COX) and inhibit respiration. Mitochondria had a very high capacity for NO scavenging, importantly, independent of NO binding to COX. NO is also known to quickly react with reactive oxygen species (ROS) in vitro. Stimulation of NO scavenging with antimycin and its inhibition by substrate depletion are consistent with NO interacting with ROS generated in Complex I or III under aerobic conditions. Extrapolating these in vitro data to the intact heart supports the hypothesis that mitochondria are a major site of cardiac NO scavenging. KEY POINTS: Cardiomyocyte scavenging of vascular nitric oxide (NO) is critical in maintaining normal cardiac function. Myoglobin redox cycling via myoglobin reductase has been proposed as a major NO scavenging site in the heart. Non-invasive optical spectroscopy was used to monitor the effect of NO on mitochondria and myoglobin redox state in intact beating heart and isolated mitochondria. These non-invasive studies reveal myoglobin/myoglobin reductase plays a minor role in cardiac NO scavenging. A high capacity for NO scavenging by heart mitochondria was demonstrated, independent of cytochrome oxidase binding but dependent on oxygen and high redox potentials consistent with generation of reactive oxygen species.

Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria.

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a3-CuB binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a3 (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420-450 nm), alpha (560-630 nm) and red (630-700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (ΔΨm) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨm-dependent, out of equilibrium, and therefore much slower than the ΔΨm-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.

Evaluation of Hepatic Iron Overload Using a Contemporary 0.55 T MRI System.

BACKGROUND: MRI T2* and R2* mapping have gained clinical acceptance for noninvasive assessment of iron overload. Lower field MRI may offer increased measurement dynamic range in patients with high iron concentration and may potentially increase MRI accessibility, but it is compromised by lower signal-to-noise ratio that reduces measurement precision. PURPOSE: To characterize a high-performance 0.55 T MRI system for evaluating patients with liver iron overload. STUDY TYPE: Prospective. POPULATION: Forty patients with known or suspected iron overload (sickle cell anemia [n = 5], ß-thalassemia [n = 3], and hereditary spherocytosis [n = 2]) and a liver iron phantom. FIELD STRENGTH/SEQUENCE: A breath-held multiecho gradient echo sequence at 0.55 T and 1.5 T. ASSESSMENT: Patients were imaged with T2*/R2* mapping 0.55 T and 1.5 T within 24 hours, and 16 patients returned for follow-up exams within 6-16 months, resulting in 56 paired studies. Liver T2* and R2* measurements and standard deviations were compared between 0.55 T and 1.5 T and used to validate a predictive model between field strengths. The model was then used to classify iron overload at 0.55 T. STATISTICAL TESTS: Linear regression and Bland-Altman analysis were used for comparisons, and measurement precision was assessed using the coefficient of variation. A P-value < 0.05 was considered statistically significant. RESULTS: R2* was significantly lower at 0.55 T in our cohort (488 ± 449 s-1 at 1.5 T vs. 178 ± 155 s-1 at 0.55 T, n = 56 studies) and in the patients with severe iron overload (937 ± 369 s-1 at 1.5 T vs. 339 ± 127 s-1 at 0.55 T, n = 23 studies). The coefficient of variation indicated reduced precision at 0.55 T (3.5 ± 2.2% at 1.5 T vs 6.9 ± 3.9% at 0.55 T). The predictive model accurately predicted 1.5 T R2* from 0.55 T R2* (Bland Altman bias = -6.6 ± 20.5%). Using this model, iron overload at 0.55 T was classified as: severe R2* > 185 s-1 , moderate 81 s-1  < R2* < 185 s-1 , and mild 45 s-1  < R2* < 91 s-1 . DATA CONCLUSION: We demonstrated that 0.55 T provides T2* and R2* maps that can be used for the assessment of liver iron overload in patients. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

Energy homeostasis is a conserved process: Evidence from Paracoccus denitrificans' response to acute changes in energy demand.

Paracoccus denitrificans is a model organism for the study of oxidative phosphorylation. We demonstrate a very high respiratory capacity compared to mitochondria when normalizing to cytochrome aa3 content even in the absence of alternative terminal oxidases. To gain insight into conserved mechanisms of energy homeostasis, we characterized the metabolic response to K+ reintroduction. A rapid 3-4-fold increase in respiration occurred before substantial cellular K+ accumulation followed by a sustained increase of up to 6-fold that persisted after net K+ uptake stopped. Proton motive force (Δp) was slightly higher upon addition of K+ with ΔpH increasing and compensating for membrane potential (ΔΨ) depolarization. Blocking the F0F1-ATP synthase (Complex V) with venturicidin revealed that the initial K+-dependent respiratory activation was primarily due to K+ influx. However, the ability to sustain an increased respiration rate was partially dependent on Complex V activity. The 6-fold stimulation of respiration by K+ resulted in a small net reduction of most cytochromes, different from the pattern observed with chemical uncoupling and consistent with balanced input and utilization of reducing equivalents. Metabolomics showed increases in glycolytic and TCA cycle intermediates together with a decrease in basic amino acids, suggesting an increased nitrogen mobilization upon K+ replenishment. ATP and GTP concentrations increased after K+ addition, indicating a net increase in cellular potential energy. Thus, K+ stimulates energy generation and utilization resulting in an almost constant Δp and increased high-energy phosphates during large acute and steady state changes in respiration. The specific energy consuming processes and signaling events associated with this simultaneous activation of work and metabolism in P. denitrificans remain unknown. Nevertheless, this homeostatic behavior is very similar to that observed in mitochondria in tissues when cellular energy requirements increase. We conclude that the regulation of energy generation and utilization to maintain homeostasis is conserved across the prokaryote/eukaryote boundary.

Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs.

Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that northern blotting of small RNAs is biased against short sequences and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm Caenorhabditis elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using northern blotting during these studies, we discovered that miRNA-length probes can have ∼1000-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable nearly equivalent detection of 24 to 14 nt RNAs. Our protocol can discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans and pRNAs in total RNA from bacteria. This improved northern blotting is particularly useful to analyze products of RNA processing or turnover, and functional RNAs that are shorter than typical miRNAs.