J. Gordon Lindsay

Heme-heme interaction in cytochrome oxidase

Abstract The cytochromes a and a 3 of intact pigeon heart mitochondria are shown to contribute equally to the α band of cytochrome oxidase in the absence of added ligands. The oxidation-reduction midpoint potentials of the cytochromes a and a 3 at pH 7·2 are −220 mV and +380 mV, respectively. The oxidation of cytochrome a 3 but not cytochrome a is coupled to a proton dissociation from a group with a p K of 7. Strong heme-heme interaction between these two cytochromes is expressed in the interdependence of their absorption spectra and half-reduction potentials. An increase of more than 50 % in the extinction coefficient of reduced cytochrome a occurs when reduced cytochrome a 3 binds CO and a similar increase in the extinction coefficient of reduced cytochrome a 3 occurs when oxidized cytochrome a binds azide. The total absorbance change on reduction of both cytochromes is unchanged by the addition of azide. Heme-heme interaction is also observed in a 30-mV increase in the half-reduction potential of cytochrome a when the measurements are carried out in the presence of CO and a 30-mV decrease in the half-reduction potential of cytochrome a 3 when the measurements are carried out in the presence of high concentrations of azide.

Metabolic Impairment Induces Oxidative Stress, Compromises Inflammatory Responses, and Inactivates a Key Mitochondrial Enzyme in Microglia

Abstract: Microglial activation, oxidative stress, and dysfunctions in mitochondria, including the reduction of cytochrome oxidase activity, have been implicated in neurodegeneration. The current experiments tested the effects of reducing cytochrome oxidase activity on the ability of microglia to respond to inflammatory insults. Inhibition of cytochrome oxidase by azide reduced oxygen consumption and increased reactive oxygen species (ROS) production but did not affect cell viability. Azide also attenuated microglial activation, as measured by nitric oxide (NO*) production in response to lipopolysaccharide (LPS). It is surprising that the inhibition of cytochrome oxidase also diminished the activity of the α‐ketoglutarate dehydrogenase complex (KGDHC), a Krebs cycle enzyme. This reduction was exaggerated when the azide‐treated microglia were also treated with LPS. The combination of the azide‐stimulated ROS and LPS‐induced NO* would likely cause peroxynitrite formation in microglia. Thus, the possibility that KGDHC was inactivated by peroxynitrite was tested. Peroxynitrite inhibited the activity of isolated KGDHC, nitrated tyrosine residues of all three KGDHC subunits, and reduced immunoreactivity to antibodies against two KGDHC components. Thus, our data suggest that inhibition of the mitochondrial respiratory chain diminishes aerobic energy metabolism, interferes with microglial inflammatory responses, and compromises mitochondrial function, including KGDHC activity, which is vulnerable to NO* and peroxynitrite that result from microglial activation. Thus, activation of metabolically compromised microglia can further diminish their oxidative capacity, creating a deleterious spiral that may contribute to neurodegeneration.

The isolation and partial characterisation of the light-harvesting pigment-protein complement of Rhodopseudomonas acidophila

The photosynthetic membranes of two strains of Rhodopseudomonas acidophila (7750 and 7050) have been resolved into their constituent light-harvesting pigment-protein complexes. Four different types of antenna complexes (B880, B800–830 and two types of B800–850) have been isolated and partially purified. In each case the light-harvesting pigments (bacteriochlorophyll a and carotenoids) are bound to rather low molecular weight polypeptides (in the 5000–9000 region).

Mitochondrial impairment in the cerebellum of the patients with progressive supranuclear palsy

Abnormalities in energy metabolism and oxidative stress accompany many neurodegenerative diseases, including progressive supranuclear palsy (PSP). Previously, we showed decreased activities of a mitochondrial enzyme complex, α‐ketoglutarate dehydrogenase complex (KGDHC), and marked increases in tissue malondialdehyde levels in post‐mortem superior frontal cortex from the patients with PSP. The current study demonstrates that KGDHC is also significantly diminished (−58%) in the cerebellum from patients with PSP (n = 14), compared to age‐matched control brains (n = 13). In contrast to cortex, markers of oxidative stress, such as malondialdehyde, tyrosine nitration or general protein carbonyl modification, did not increase in cerebellum. Furthermore, the protein levels of the individual components of KGDHC did not decline. The activities of two other mitochondrial enzymes were measured to determine whether the changes in KGDHC were selective. The activity of aconitase, a mitochondrial enzyme with an iron/sulfur cluster, is also significantly diminished (−50%), whereas glutamate dehydrogenase activity is unchanged. The present results suggest that the interaction of metabolic impairment and oxidative stress is region‐specific in PSP brain. In cerebellum, reductions in KGDHC occur in the absence of increases in common measures of oxidative stress, and may underlie the metabolic deficits and contribute to pathological and clinical manifestation related to the cerebellum in patients with PSP.

A further characterisation of the B890 light-harvesting pigment—protein complex from Rhodospirillum rubrum strain S1

An intact B890 light‐harvesting pigment—protein complex has been obtained from Rhodospirillum rubrum strain S1. We show that this complex contains two types of low‐M r polypeptide. Both these polypeptides are present in the intact chromatophore membrane. Analysis of the pigment content of this complex suggests that per pair of polypeptides the complex contains 2 molecules of bacteriochlorophyll and one molecule of carotenoid.

Immunochemical Characterization of the Deficiency of the α‐Ketoglutarate Dehydrogenase Complex in Thiamine‐Deficient Rat Brain

Abstract: Mitochondrial dysfunction is a common feature of many neurodegenerative disorders. The metabolic encephalopathy caused by thiamine deficiency (TD) is a classic example in which an impairment of cerebral oxidative metabolism leads to selective cell death. In experimental TD in rodents, a reduction in the activity of the thiamine diphosphate‐dependent, mitochondrial enzyme α‐ketoglutarate dehydrogenase complex (KGDHC) occurs before the onset of pathologic lesions and is among the earliest biochemical deficits found. To understand the molecular basis and the significance of the deficiency of KGDHC in TD‐induced brain damage, the enzyme activity and protein levels of KGDHC were analyzed. The effect of TD on the subregional/cellular distribution of KGDHC and the anatomic relation of KGDHC with selective cell death were also tested by immunocytochemistry. Consistent with several previous studies, TD dramatically reduced KGDHC activity in both anatomically damaged (thalamus and inferior colliculus) and spared (cerebral cortex) regions. Immunocytochemistry revealed no apparent correlation of regional KGDHC immunoreactivity or its response to TD with affected regions in TD. The basis of the enzymatic and immunocytochemical behavior of KGDHC was further assessed by quantitative immunoblots, using antibodies specific for each of the three KGDHC components. Despite the marked decrease of KGDHC activity in TD, no reduction of any of the three KGDHC protein levels was found. Thus, TD impairs the efficacy of the KGDHC catalytic machinery, whereas the concentration of protein molecules persists. The generalized decline of KGDHC activity with no apparent anatomic selectivity is consistent with the notion that the compromised mitochondrial oxidation sensitizes the brain cells to various other insults that precipitate the cell death. The current TD model provides a relevant experimental system to understand the molecular basis of many neurodegenerative conditions in which mitochondrial dysfunction and KGDHC deficiency are prominent features.

Cellular mitochondrial heterogeneity in cultured astrocytes as demonstrated by immunogold labeling of α-ketoglutarate dehydrogenase

In brain cells, various metabolites and metabolic pathways, largely of mitochondrial origin, have been shown to be compartmentalized. Attention has therefore been focused on the possible existence of mitochondrial heterogeneity in the brain at the cellular level. To determine whether mitochondria in cultured cortical and cerebellar astrocytes are heterogeneous at the single cell level, immunogold electron microscopy and an antibody against the α‐ketoglutarate dehydrogenase component of the α‐ketoglutarate dehydrogenase complex, a marker enzyme for the tricarboxylic acid (TCA) cycle, were employed. The number of gold particles was counted in the mitochondria of 36 and 42 cells from cultured cerebellar and cortical astrocytes, respectively. A test for random distribution (Poisson distribution) of mitochondria according to the number of gold particles was subsequently performed for every one of the 36 and 42 cells as the ratio variance/mean (= index of dispersion). This should be approximately distributed as χ2/degrees of freedom (df) = n − 1, n = number of mitochondria), if the observations obeyed a Poisson distribution. For 26 of the 36 (cerebellar astrocytes) distributions and for 28 of the 42 (cortical astrocytes) distributions a random distribution had to be rejected. These findings therefore strongly indicate that α‐ketoglutarate dehydrogenase is heterogeneously distributed in mitochondria within individual astrocytes originating either from cerebellum or cerebral cortex. In conclusion, this study underlines the probability that mitochondrial heterogeneity at the single cell level might be extended to involve other metabolic pathways and metabolites.

Structure-Function Analysis of Recombinant Substrate Protein 22 kDa (SP-22) A MITOCHONDRIAL 2-CYS PEROXIREDOXIN ORGANIZED AS A DECAMERIC TOROID

Bovine mitochondrial SP-22 is a member of the peroxiredoxin family of peroxidases. It belongs to the peroxiredoxin 2-Cys subgroup containing three cysteines at positions 47, 66, and 168. The cloning and overexpression in Escherichia coli of recombinant wild type SP-22 and its three cysteine mutants (C47S, C66S, and C168S) are reported. Purified His-tagged SP-22 was fully active with Cys-47 being confirmed as the catalytic residue. The enzyme forms a stable decameric toroid consisting of five basic dimeric units containing intermolecular disulfide bonds linking the catalytically active Cys-47 of one subunit and Cys-168 of the adjacent monomer. The disulfide bonds are not required for overall structural integrity. The toroidal units have average external and internal diameters of 15 and 7 nm, respectively, and can form stacks in a lateral arrangement of two or three rings. C47S had a pronounced tendency to stack in long tubular structures containing up to 60 rings. Further unusual structural features are the presence of radial spikes projecting from the external surface and ordered electron-dense material within the central cavity of the toroid.

Structural and enzymological analysis of the interaction of isolated domains of cytochrome P-450 BM3.

The interactions of the individually expressed haem‐ and flavin‐containing domains of cytochrome P‐450 BM3 have been analysed by enzymological and spectroscopic techniques. Electron transfer between the isolated domains occurs at a much lower rate than that occurring in the intact flavocytochrome. CD spectroscopic studies indicate that the linkage of the domains in intact P‐450 BM3 creates haem and amino acid environments suitable for efficient electron transfer from its flavin domain.

The identification of T2; the phosphate/pyrophosphate transport protein of the hepatic microsomal glucose-6-phosphatase system.

The phosphate/pyrophosphate translocase protein (T2) of the hepatic microsomal glucose‐6‐phosphatase system was identified and then purified using antibodies raised against the rat mitochondrial phosphate/hydroxyl ion antiport protein. The T2 protein was shown to be absent in the microsomes isolated from a patient previously diagnosed as having type 1c glycogen storage disease.