Fasting and fasting-mimicking treatment activate SIRT1/LXRα and alleviate diabetes-induced systemic and microvascular dysfunction.

AIMS/HYPOTHESIS: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage. METHODS: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis. RESULTS: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression. CONCLUSIONS/INTERPRETATION: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.

Assessment of the Maximal Activity of Complex IV in the Inner Mitochondrial Membrane by Tandem Electrochemistry and Respirometry.

Assessment of activities of mitochondrial electron transport enzymes is important for understanding mechanisms of metabolic diseases, but structural organization of mitochondria and low sample availability pose distinctive challenges for in situ functional studies. We report the development of a tandem microfluidic respirometer that simultaneously tracks both the reduction of mediators on the electrode and the ensuing reduction of O2 by complex IV in the inner mitochondrial membrane. The response time of O2 consumption to multiple alternating potential steps is of approximately 10 s for a 150 µm-thick sample. Steady O2 depletion shows good quantitative correlation with the supplied electric charge, Pearson's r = 0.994. Reduction of mediators on biocompatible gold electrodes modified with carbon ink or fumed silica can compete with the oxidation of mediators by mitochondria, yielding an overall respiratory activity comparable to that upon chemical reduction by ascorbate. The dependence of O2 consumption on mediator and mitochondrial suspension concentrations shows that mass transport between the electrode and mitochondria does not limit biological activity of the latter. The mediated electrochemical approach is validated by the radiometric measurements of simulated changes in the intrinsic mitochondrial activity upon partial inhibition of complex IV by NaN3. This approach enables the development of O2-independent, biomimetic electrochemical assays narrowly targeting components of the electron transport chains in their native environments.

Iron-Containing Ureases.

Conventional ureases possess dinuclear nickel active sites that are oxygen-stable and require a set of accessory proteins for metallocenter biosynthesis. By contrast, oxygen-labile ureases have active sites containing dual ferrous ions and lack a requirement for maturation proteins. The structures of the two types of urease are remarkably similar, with an active site architecture that includes two imidazoles and a carboxylate ligand coordinated to one metal, two imidazoles coordinated to the second metal, and a metal-bridging carbamylated lysine ligand. The electronic spectrum of the diferric form of the enzyme resembles that of methemerythrin. Resonance Raman spectroscopic analyses confirm the presence of a µ-oxo ligand and indicate the presence of one or more terminal solvent ligands.

Mitochondrial Ceramide Effects on the Retinal Pigment Epithelium in Diabetes.

Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood-retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1ß, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 µM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Structural Origin of the Large Redox-Linked Reorganization in the 2-Oxoglutarate Dependent Oxygenase, TauD.

2-Oxoglutarate (2OG)-dependent oxygenases catalyze a wide range of chemical transformations via C-H bond activation. Prior studies raised the question of whether substrate hydroxylation by these enzymes occurs via a hydroxyl rebound or alkoxide mechanism and highlighted the need to understand the thermodynamic properties of transient intermediates. A recent spectroelectrochemical investigation of the 2OG-dependent oxygenase, taurine hydroxylase (TauD), revealed a strong link between the redox potential of the Fe(II)/Fe(III) couple and conformational changes of the enzyme. In this study, we show that the redox potential of wild-type TauD varies by 468 mV between the reduction of 2OG-Fe(III)-TauD (-272 mV) and oxidation of 2OG-Fe(II)-TauD (+196 mV). We use active site variants to investigate the structural origin of the redox-linked reorganization and the contributions of the metal-bound residues to the dynamic tuning of the redox potential of TauD. Time-dependent redox titrations show that reorganization occurs as a multistep process. Transient optical absorption and infrared spectroelectrochemistry show that substitution of any metal ligand alters the kinetics and thermodynamics of the reorganization. The H99A variant shows the largest net redox change relative to the wild-type protein, suggesting that redox-coupled protonation of H99 is required for high redox potentials of the metal. The D101Q and H255Q variants also suppress the conformational change, supporting their involvement in the structural rearrangement. Similar redox-linked conformational changes are observed in another 2OG dependent oxygenase, ethylene-forming enzyme, indicating that dynamic structural flexibility and the associated thermodynamic tuning may be a common phenomenon in this family of enzymes.

Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD.

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an in situ structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semiempirical computational methods, demonstrating that the Fe(III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and +171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox-difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

Fourier Transform Infrared Spectrovoltammetry and Quantitative Modeling of Analytes in Kinetically Constrained Redox Mixtures.

Redox-active analytes that do not support direct electron transfer on the electrode, such as proteins with buried redox centers, pose challenges to characterization of their structural and thermodynamic properties. Investigations of indirect transitions in analytes supported by complex redox mixtures require a careful balance between kinetic limitations and spectral interference from the mediators. Using methylene green and thionine acetate as redox mediators and myoglobin as the analyte, we demonstrate that normal pulse spectrovoltammetry (NPSV) with Fourier transform infrared (FT-IR) detection and subsequent global spectral regression analysis can resolve structural and thermodynamic properties simultaneously with little a priori information. Both the E1/2 and unbiased redox difference FT-IR spectra of the Fe(II)/Fe(III) redox couple of myoglobin in reduction and oxidation NPSV modes were in good agreement with those reported earlier by independent techniques. The thermodynamic and kinetic limitations of mediators/analyte interactions were investigated using comprehensive semiempirical kinetic simulation models. This modeling effort yielded a flexible computational tool capable of quantitatively predicting the redox response in mediated electrochemical studies and defining its limitations, thus greatly expanding the range and precision of the formal mediator/analyte concentration ratio rule.

Micro-respirometry of whole cells and isolated mitochondria.

Oxygen consumption is a key metric of metabolism in aerobic organisms. Current respirometric methods led to seminal discoveries despite limitations such as high sample demand, exchange with atmospheric O2, and cumulative titration protocols leading to limited choice of useable tissue, complex data interpretation, and restricted experimental design. We developed a sensitive and customizable method of measuring O2 consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment. We demonstrate that O2 permeability of the photopolymer, VeroClear, is comparable to that of polyetheretherketone (0.125 vs. 0.143 barrer, respectively) providing an efficient barrier to oxygen ingress. Optical transparency of VeroClear, combined with high resolution 3D printing, allows for optode-based oxygen detection in enclosed samples. These properties yield a microrespirometer with over 100× dynamic range for O2 consumption rates. Importantly, the enclosed respirometer configuration and very low oxygen permeability of materials makes it suitable, with resin pre-conditioning, for quantitative assessment of O2 consumption rates at any desired [O2], including hyperbaric, physiological or hypoxic conditions as necessary for each cell type. We characterized two configurations to study soluble enzymes, isolated mitochondria, cells in suspension, and adherent cells cultured on-chip. Improved sensitivity allows for routine quantitative detection of respiration by as few as several hundred cells. Specific activity of cell suspensions in the microrespirometer was in close agreement with that obtained by high-resolution polarographic respirometry. Adherent cell protocols allowed for physiologically relevant assessment of respiration in retinal pigment epithelial cells, ARPE-19, which displayed lower metabolic rates compared with those in suspension. By exchanging medium composition, we demonstrate that cells can be transiently inhibited by cyanide and that 99.6% of basal O2 uptake is recovered upon its removal. This approach is amenable to new experimental designs and precision measurements on limited sample quantities across basic research and applied fields.

Spectroscopic analyses of 2-oxoglutarate-dependent oxygenases: TauD as a case study.

A wide range of spectroscopic approaches have been used to interrogate the mononuclear iron metallocenter in 2-oxoglutarate (2OG)-dependent oxygenases. The results from these spectroscopic studies have provided valuable insights into the structural changes at the active site during substrate binding and catalysis, thus providing critical information that complements investigations of these enzymes by X-ray crystallography, biochemical, and computational approaches. This mini-review highlights taurine hydroxylase (taurine:2OG dioxygenase, TauD) as a case study to illustrate the wealth of knowledge that can be generated by applying a diverse array of spectroscopic investigations to a single enzyme. In particular, electronic absorption, circular dichroism, magnetic circular dichroism, conventional and pulse electron paramagnetic, Mössbauer, X-ray absorption, and resonance Raman methods have been exploited to uncover the properties of the metal site in TauD.

Effects of Film Morphology and Surface Chemistry on the Direct Electrochemistry of Cytochrome c at Boron-Doped Diamond Electrodes.

The effects of film morphology and surface termination on the direct electron transfer of horse heart cytochrome c on boron-doped ultrananocrystalline (B-UNCD) and microcrystalline (B-MCD) diamond thin-film electrodes were investigated. Quasi-reversible, diffusion-controlled cyclic voltammetric responses were observed on oxygen-terminated (atomic O/C ~0.015), but not hydrogen-terminated (atomic O/C ~0.02) diamond thin films. The effect of the surface termination was the same for both the nanostructured B-UNCD film with sp2-bonded carbon atoms in the grain boundaries and the well faceted B-MCD film with micron-sized grains and largely devoid of sp2 carbon. Stable cyclic voltammetric i-E curves were recorded with cycling for both oxygen-terminated films indicating the absence of protein denaturation and electrode fouling. The peak currents increased linearly with the square root of the scan rate and the protein concentration; both indicative of a reaction rate limited by semi-infinite linear diffusion of the protein. Similar heterogeneous electron-transfer rate constants were observed for oxygen-terminated B-UNCD (3.48 (± 1.25) × 10-3 cm/s) and B-MCD films (2.38 (± 0.72) × 10-3 cm/s). The results clearly reveal that the oxygen-terminated surface is more active for electron-transfer with this soluble redox protein than is the hydrogen-terminated surface. The film morphology does not influence the diffusion-controlled response of the redox protein.

Structure of the key species in the enzymatic oxidation of methane to methanol.

Methane monooxygenase (MMO) catalyses the O2-dependent conversion of methane to methanol in methanotrophic bacteria, thereby preventing the atmospheric egress of approximately one billion tons of this potent greenhouse gas annually. The key reaction cycle intermediate of the soluble form of MMO (sMMO) is termed compound Q (Q). Q contains a unique dinuclear Fe(IV) cluster that reacts with methane to break an exceptionally strong 105 kcal mol(-1) C-H bond and insert one oxygen atom. No other biological oxidant, except that found in the particulate form of MMO, is capable of such catalysis. The structure of Q remains controversial despite numerous spectroscopic, computational and synthetic model studies. A definitive structural assignment can be made from resonance Raman vibrational spectroscopy but, despite efforts over the past two decades, no vibrational spectrum of Q has yet been obtained. Here we report the core structures of Q and the following product complex, compound T, using time-resolved resonance Raman spectroscopy (TR(3)). TR(3) permits fingerprinting of intermediates by their unique vibrational signatures through extended signal averaging for short-lived species. We report unambiguous evidence that Q possesses a bis-µ-oxo diamond core structure and show that both bridging oxygens originate from O2. This observation strongly supports a homolytic mechanism for O-O bond cleavage. We also show that T retains a single oxygen atom from O2 as a bridging ligand, while the other oxygen atom is incorporated into the product. Capture of the extreme oxidizing potential of Q is of great contemporary interest for bioremediation and the development of synthetic approaches to methane-based alternative fuels and chemical industry feedstocks. Insight into the formation and reactivity of Q from the structure reported here is an important step towards harnessing this potential.

Apoprotein isolation and activation, and vibrational structure of the Helicobacter mustelae iron urease.

The micro aerophilic pathogen Helicobacter mustelae synthesizes an oxygen-labile, iron-containing urease (UreA2B2) in addition to its standard nickel-containing enzyme (UreAB). An apoprotein form of the iron urease was prepared from ureA2B2-expressing recombinant Escherichia coli cells that were grown in minimal medium. Temperature-dependent circular dichroism measurements of holoprotein and apoprotein demonstrate an enhancement of thermal stability associated with the UreA2B2 metallocenter. In parallel to the situation reported for nickel activation of the standard urease apoprotein, incubation of UreA2B2 apoprotein with ferrous ions and bicarbonate generated urease activity in a portion of the nascent active sites. In addition, ferrous ions were shown to be capable of reductively activating the oxidized metallocenter. Resonance Raman spectra of the inactive, aerobically-purified UreA2B2 holoprotein exhibit vibrations at 495cm(-1) and 784cm(-1), consistent with ν(s) and ν(as) modes of an Fe(III)OFe(III) center; these modes undergo downshifts upon binding of urea and were unaffected by changes in pH. The low-frequency mode also exhibits an isotopic shift from 497 to 476cm(-1) upon (16)O/(18)O bulk water isotope substitution. Expression of subunits of the conventional nickel-containing Klebsiella aerogenes urease in cells grown in rich medium without nickel resulted in iron incorporation into a portion of the protein. The inactive iron-loaded species exhibited a UV-visible spectrum similar to oxidized UreA2B2 and was capable of being reductively activated under anoxic conditions. Results from these studies more clearly define the formation and unique properties of the iron urease metallocenter.

Equilibrium and kinetic behavior of Fe(CN)6(3-/4-) and cytochrome c in direct electrochemistry using a film electrode thin-layer transmission cell.

We report on the design and performance of a thin-layer electrochemical cell optimized for use with optically transparent film electrodes in combination with UV/vis and IR transmission spectroscopic measurements. The cell allows for measurements under both aerobic and anaerobic conditions. The direct, unmediated electron transfer, as assessed by the current transient, and the corresponding optical response observed for the Fe(CN)(6)(3-/4-) couple were in good agreement with theoretical predictions for voltammetry and optical absorption by an analyte confined in a thin layer. Chronoamperometric and spectroscopic measurements of Fe(CN)(6)(3-/4-) on gold mesh electrode revealed fast kinetics strongly influenced by the electrolyte concentration. Maximal apparent rates exceeding 2 s(-1) in 1 M KCl were observed optically. The direct kinetic and thermodynamic behavior of cytochrome c was compared with several electrode materials using the cell. The results showed heme ligand-dependent changes in the protein-electrode interactions. Mid-UV/visible spectral changes upon redox transitions in native cytochrome c and its cyanide derivative, as well as dissociation of the ferrous cytochrome c-CN complex, are reported.

Insight into the mechanism of an iron dioxygenase by resolution of steps following the FeIV=HO species.

Iron oxygenases generate elusive transient oxygen species to catalyze substrate oxygenation in a wide range of metabolic processes. Here we resolve the reaction sequence and structures of such intermediates for the archetypal non-heme Fe(II) and alpha-ketoglutarate-dependent dioxygenase TauD. Time-resolved Raman spectra of the initial species with (16)O(18)O oxygen unequivocally establish the Fe(IV) horizontal lineO structure. (1)H/(2)H substitution reveals direct interaction between the oxo group and the C1 proton of substrate taurine. Two new transient species were resolved following Fe(IV) horizontal lineO; one is assigned to the nu(FeO) mode of an Fe(III) horizontal line O(H) species, and a second is likely to arise from the vibration of a metal-coordinated oxygenated product, such as Fe(II) horizontal line O horizontal line C(1) or Fe(II) horizontal line OOCR. These results provide direct insight into the mechanism of substrate oxygenation and suggest an alternative to the hydroxyl radical rebinding paradigm.

Metal and substrate binding to an Fe(II) dioxygenase resolved by UV spectroscopy with global regression analysis.

The addition of divalent metal ions or substrate taurine to TauD, an alpha-ketoglutarate-dependent dioxygenase, alters its UV absorption, as clearly observed by monitoring the protein's difference spectra. Binding of metal ions leads to a decrease in absorption at approximately 297 nm and modulation of other features. A separate signature with enhanced absorption at approximately 295 nm is identified for binding of taurine. These narrow ( approximately 700 cm(-1)) and intense ( approximately 0.5mM(-1) cm(-1)) spectral changes are attributed to ligand-induced protein conformational changes affecting the environment of aromatic residues. The changes in the UV difference spectra were exploited to assess directly the thermodynamics and kinetics of ligand interactions in wild-type TauD and selected variants. This approach holds promise as a new tool to probe ligand-induced conformational changes in a wide range of other proteins. Experimental and quantification approaches for a reliable analysis of protein absorption below 320 nm are presented.

Redox-dependent conformational changes in cytochrome C oxidase suggest a gating mechanism for proton uptake.

A role for conformational change in the coupling mechanism of cytochrome c oxidase is the subject of controversy. Relatively small conformational changes have been reported in comparisons of reduced and oxidized crystal structures of bovine oxidase but none in bacterial oxidases. Comparing the X-ray crystal structures of the reduced (at 2.15 A resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, we observe a displacement of heme a(3) involving both the porphyrin ring and the hydroxyl farnesyl tail, accompanied by protein movements in nearby regions, including the mid part of helix VIII of subunit I which harbors key residues of the K proton uptake path, K362 and T359. The conformational changes in the reduced form are reversible upon reoxidation. They result in an opening of the top of the K pathway and more ordered waters being resolved in that region, suggesting an access path for protons into the active site. In all high-resolution structures of oxidized R. sphaeroides cytochrome c oxidase, a water molecule is observed in the hydrophobic region above the top of the D path, strategically positioned to facilitate the connection of residue E286 of subunit I to the active site or to the proton pumping exit path. In the reduced and reduced plus cyanide structures, this water molecule disappears, implying disruption of proton conduction from the D path under conditions when the K path is open, thus providing a mechanism for alternating access to the active site.

Optically transparent diamond electrode for use in ir transmission spectroelectrochemical measurements.

A new analytical spectroelectrochemical methodology is reported on that utilizes an optically transparent boron-doped diamond thin film. The film was deposited on undoped Si by microwave-assisted chemical vapor deposition using a 4-h growth with a 0.5% CH4/H2 source gas mixture and 2 ppm B2H6 added for boron doping. The thin-film electrode possessed a transparency of 40-60% in the mid- and far-IR regions of the electromagnetic spectrum. The physical, electrical, optical, and electrochemical properties of the electrode were characterized by scanning electron microscopy, Raman spectroscopy, X-ray diffraction, four-point probe electrical resistance measurements, IR spectroscopy, and cyclic voltammetry. The film's electrochemical behavior was evaluated using both aqueous (Fe(CN)(6)3-/4-, methyl viologen, Ru(NH3)(6)3+/2+, and IrCl(6)2-/3-) and nonaqueous (ferrocene) redox systems. The film exhibited a low and stable background current and a nearly reversible voltammetric response for all these redox systems. The diamond/Si optically transparent electrode (OTE) and a thin-layer transmission cell were used to record the spectroelectrochemical response for 10 mM Fe(CN)(6)3-/4- in 1 M KCl. Difference IR spectra (oxidized minus reduced), recorded at various applied potentials, showed that the CN vibrational mode at 2039 cm-1 for Fe(CN)(6)4- reversibly shifted to 2116 cm-1 upon oxidation to Fe(CN)(6)3-, as expected. Difference IR spectra (oxidized minus reduced) were also recorded for 20 mM ferrocene in 0.1 M TBABF4/CH3CN. A shift of the C-H bending mode of the cyclopentadienyl ring from 823 to 857 cm-1 occurred upon oxidation of ferrocene to ferricenium. The key finding from the work is that the diamond OTE provides sensitive, reproducible, and stable spectroelectrochemical responses for aqueous and nonaqueous redox systems in the mid- and far-IR.

UV optical absorption by protein radicals in cytochrome c oxidase.

The UV properties of key oxygen intermediates of cytochrome c oxidase have been investigated by transient absorption spectroscopy. The temporal behavior of P(m) species upon aerobic incubation with CO or in the reaction with H(2)O(2) is closely concurred by a new optical shift at 290/260 nm. In the acid-induced conversion of P(m) to F(*), it is replaced by another shift at 323/288 nm. The wavelength and intensity of the UV signal observed in F(*) match closely the properties of model Trp? in agreement with results of ENDOR studies on this species. The UV spectrum of Tyr* gives the closest match with the 290/260 nm signal observed in P(m). On the basis of analysis of possible UV chromophores in CcO and similarity to Tyr*, the 290/260 nm signal is proposed to originate from the H(240)-Y(244)* site. Possible effects of local environment on UV properties of this site are discussed.