Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin: implications for blood substitute design.

Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic heme-mediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the ß-subunit (F41). We therefore replaced this residue with a tyrosine (ßF41Y, Hb Mequon). The ßF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, ßF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the ß-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in ßF41Y. NO bioavailability was enhanced in ßF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the ß-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.

Hemoglobin Effects on Nitric Oxide Mediated Hypoxic Vasodilation.

The brain responds to hypoxia with an increase in cerebral blood flow (CBF). However, such an increase is generally believed to start only after the oxygen tension decreases to a certain threshold level. Although many mechanisms (different vasodilator and different generation and metabolism mechanisms of the vasodilator) have been proposed at the molecular level, none of them has gained universal acceptance. Nitric oxide (NO) has been proposed to play a central role in the regulation of oxygen supply since it is a vasodilator whose production and metabolism are both oxygen dependent. We have used a computational model that simulates blood flow and oxygen metabolism in the brain (BRAINSIGNALS) to test mechanism by which NO may elucidate hypoxic vasodilation. The first model proposed that NO was produced by the enzyme nitric oxide synthase (NOS) and metabolized by the mitochondrial enzyme cytochrome c oxidase (CCO). NO production declined with decreasing oxygen concentration given that oxygen is a substrate for nitric oxide synthase (NOS). However, this was balanced by NO metabolism by CCO, which also declined with decreasing oxygen concentration. However, the NOS effect was dominant; the resulting model profiles of hypoxic vasodilation only approximated the experimental curves when an unfeasibly low K m for oxygen for NOS was input into the model. We therefore modified the model such that NO generation was via the nitrite reductase activity of deoxyhemoglobin instead of NOS, whilst keeping the metabolism of NO by CCO the same. NO production increased with decreasing oxygen concentration, leading to an improved reproduction of the experimental CBF versus PaO2 curve. However, the threshold phenomenon was not perfectly reproduced. In this present work, we incorporated a wider variety of oxygen dependent and independent NO production and removal mechanisms. We found that the addition of NO removal via oxidation to nitrate mediated by oxyhemoglobin resulted in the optimum fit of the threshold phenomenon by the model. Our revised model suggests, but does not prove, that changes in NO concentration can be the primary cause of the relationship between pO2 and cerebral blood flow.

Re-evaluation of the near infrared spectra of mitochondrial cytochrome c oxidase: Implications for non invasive in vivo monitoring of tissues.

We re-determined the near infrared (NIR) spectral signatures (650-980nm) of the different cytochrome c oxidase redox centres, in the process separating them into their component species. We confirm that the primary contributor to the oxidase NIR spectrum between 700 and 980nm is cupric CuA, which in the beef heart enzyme has a maximum at 835nm. The 655nm band characterises the fully oxidised haem a3/CuB binuclear centre; it is bleached either when one or more electrons are added to the binuclear centre or when the latter is modified by ligands. The resulting 'perturbed' binuclear centre is also characterised by a previously unreported broad 715-920nm band. The NIR spectra of certain stable liganded species (formate and CO), and the unstable oxygen reaction compounds P and F, are similar, suggesting that the latter may resemble the stable species electronically. Oxidoreduction of haem a makes no contribution either to the 835nm maximum or the 715nm band. Our results confirm the ability of NIRS to monitor the CuA centre of cytochrome oxidase activity in vivo, although noting some difficulties in precise quantitative interpretations in the presence of perturbations of the haem a3/CuB binuclear centre.

Cytochrome c oxidase response to changes in cerebral oxygen delivery in the adult brain shows higher brain-specificity than haemoglobin.

The redox state of cerebral mitochondrial cytochrome c oxidase monitored with near-infrared spectroscopy (Δ[oxCCO]) is a signal with strong potential as a non-invasive, bedside biomarker of cerebral metabolic status. We hypothesised that the higher mitochondrial density of brain compared to skin and skull would lead to evidence of brain-specificity of the Δ[oxCCO] signal when measured with a multi-distance near-infrared spectroscopy (NIRS) system. Measurements of Δ[oxCCO] as well as of concentration changes in oxygenated (Δ[HbO2]) and deoxygenated haemoglobin (Δ[HHb]) were taken at multiple source-detector distances during systemic hypoxia and hypocapnia (decrease in cerebral oxygen delivery), and hyperoxia and hypercapnia (increase in cerebral oxygen delivery) from 15 adult healthy volunteers. Increasing source-detector spacing is associated with increasing light penetration depth and thus higher sensitivity to cerebral changes. An increase in Δ[oxCCO] was observed during the challenges that increased cerebral oxygen delivery and the opposite was observed when cerebral oxygen delivery decreased. A consistent pattern of statistically significant increasing amplitude of the Δ[oxCCO] response with increasing light penetration depth was observed in all four challenges, a behaviour that was distinctly different from that of the haemoglobin chromophores, which did not show this statistically significant depth gradient. This depth-dependence of the Δ[oxCCO] signal corroborates the notion of higher concentrations of CCO being present in cerebral tissue compared to extracranial components and highlights the value of NIRS-derived Δ[oxCCO] as a brain-specific signal of cerebral metabolism, superior in this aspect to haemoglobin.

Nitrite binding to globins: linkage isomerism, EPR silence and reductive chemistry.

The nitrite adducts of globins can potentially bind via O- or N- linkage to the heme iron. We have used EPR (electron paramagnetic resonance) and DFT (density functional theory) to explore these binding modes to myoglobin and hemoglobin. We demonstrate that the nitrite adducts of both globins have detectable EPR signals; we provide an explanation for the difficulty in detecting these EPR features, based on uniaxial state considerations. The EPR and DFT data show that both nitrite linkage isomers can be present at the same time and that the two isomers are readily interconvertible in solution. The millisecond-scale process of nitrite reduction by Hb is investigated in search of the elusive Fe(II)-nitrite adduct.

The influence of carbon dioxide on cerebral metabolism and oxygen consumption: combining multimodal monitoring with dynamic systems modelling.

Hypercapnia increases cerebral blood flow. The effects on cerebral metabolism remain incompletely understood although studies show an oxidation of cytochrome c oxidase, Complex IV of the mitochondrial respiratory chain. Systems modelling was combined with previously published non-invasive measurements of cerebral tissue oxygenation, cerebral blood flow, and cytochrome c oxidase redox state to evaluate any metabolic effects of hypercapnia. Cerebral tissue oxygen saturation and cytochrome oxidase redox state were measured with broadband near infrared spectroscopy and cerebral blood flow velocity with transcranial Doppler ultrasound. Data collected during 5-min hypercapnia in awake human volunteers were analysed using a Fick model to determine changes in brain oxygen consumption and a mathematical model of cerebral hemodynamics and metabolism (BrainSignals) to inform on mechanisms. Either a decrease in metabolic substrate supply or an increase in metabolic demand modelled the cytochrome oxidation in hypercapnia. However, only the decrease in substrate supply explained both the enzyme redox state changes and the Fick-calculated drop in brain oxygen consumption. These modelled outputs are consistent with previous reports of CO2 inhibition of mitochondrial succinate dehydrogenase and isocitrate dehydrogenase. Hypercapnia may have physiologically significant effects suppressing oxidative metabolism in humans and perturbing mitochondrial signalling pathways in health and disease.

Cold-Water Immersion and Lower Limb Muscle Oxygen Consumption as Measured by Near-Infrared Spectroscopy in Trained Endurance Athletes.

CONTEXT: Cold-water immersion (CWI) has been reported to reduce tissue metabolism postimmersion, but physiological data are lacking regarding the muscle metabolic response to its application. Near-infrared spectroscopy (NIRS) is a noninvasive optical technique that can inform muscle hemodynamics and tissue metabolism. OBJECTIVE: To investigate the effects of CWI at 2 water temperatures (10°C and 15°C) on NIRS-calculated measurements of muscle oxygen consumption (mVO2). DESIGN: Crossover study. SETTING: University sports rehabilitation center. PATIENTS OR OTHER PARTICIPANTS: A total of 11 male National Collegiate Athletic Association Division II long-distance runners (age = 23.4 ± 3.4 years, height = 1.8 ± 0.1 m, mass = 68.8 ± 10.7 kg, mean adipose tissue thickness = 6.7 ± 2.7 mm). INTERVENTION(S): Cold-water immersion at 10°C and 15°C for 20 minutes. MAIN OUTCOME MEASURE(S): We calculated mVO2 preimmersion and postimmersion at water temperatures of 10°C and 15°C. Changes in tissue oxyhemoglobin (O2Hb), deoxyhemoglobin (HHb), total hemoglobin (tHb), hemoglobin difference (Hbdiff), and tissue saturation index (TSI %) were measured during the 20-minute immersion at both temperatures. RESULTS: We observed a decrease in mVO2 after immersion at both 10°C and 15°C (F1,9 = 27.7801, P = .001). During the 20-minute immersion at both temperatures, we noted a main effect of time for O2Hb (F3,27 = 14.227, P = .001), HHb (F3,27 = 5.749, P = .009), tHb (F3,27 = 24.786, P = .001), and Hbdiff (F3,27 = 3.894, P = .020), in which values decreased over the course of immersion. Post hoc pairwise comparisons showed that these changes occurred within the final 5 minutes of immersion for tHb and O2Hb. CONCLUSIONS: A 20-minute CWI at 10°C and 15°C led to a reduction in mVO2. This was greater after immersion at 10°C. The reduction in mVO2 suggests a decrease in muscle metabolic activity (ie, O2 use after CWI). Calculating mVO2 via the NIRS-occlusion technique may offer further insight into muscle metabolic responses beyond what is attainable from observing the NIRS primary signals.

Taming hemoglobin chemistry-a new hemoglobin-based oxygen carrier engineered with both decreased rates of nitric oxide scavenging and lipid oxidation.

The clinical utility of hemoglobin-based oxygen carriers (HBOC) is limited by adverse heme oxidative chemistry. A variety of tyrosine residues were inserted on the surface of the γ subunit of recombinant fetal hemoglobin to create novel electron transport pathways. This enhanced the ability of the physiological antioxidant ascorbate to reduce ferryl heme and decrease lipid peroxidation. The γL96Y mutation presented the best profile of oxidative protection unaccompanied by loss of protein stability and function. N-terminal deletions were constructed to facilitate the production of recombinant hemoglobin by fermentation and phenylalanine insertions in the heme pocket to decrease the rate of NO dioxygenation. The resultant mutant (αV1del. αL29F, γG1del. γV67F, γL96Y) significantly decreased NO scavenging and lipid peroxidation in vitro. Unlike native hemoglobin or a recombinant control (αV1del, γG1del), this mutation showed no increase in blood pressure immediately following infusion in a rat model of reperfusion injury, suggesting that it was also able to prevent NO scavenging in vivo. Infusion of the mutant also resulted in no meaningful adverse physiological effects apart from diuresis, and no increase in oxidative stress, as measured by urinary isoprostane levels. Following PEGylation via the Euro-PEG-Hb method to increase vascular retention, this novel protein construct was compared with saline in a severe rat reperfusion injury model (45% blood volume removal for 90 minutes followed by reinfusion to twice the volume of shed blood). Blood pressure and survival were followed for 4 h post-reperfusion. While there was no difference in blood pressure, the PEGylated Hb mutant significantly increased survival.

Sulfide inhibition of and metabolism by cytochrome c oxidase.

Hydrogen sulfide (H2S), a classic cytochrome c oxidase inhibitor, is also an in vitro oxidase substrate and an in vivo candidate hormonal ('gasotransmitter') species affecting sleep and hibernation. H2S, nitric oxide (NO) and carbon monoxide (CO) share some common features. All are low-molecular-mass physiological effectors and also oxidase inhibitors, capable of binding more than one enzyme site, and each is an oxidizable 'substrate'. The oxidase oxidizes CO to CO2, NO to nitrite and sulfide to probable persulfide species. Mitochondrial cytochrome c oxidase in an aerobic steady state with ascorbate and cytochrome c is rapidly inhibited by sulfide in a biphasic manner. At least two successive inhibited species are involved, probably partially reduced. The oxidized enzyme, in the absence of turnover, occurs in at least two forms: the 'pulsed' and 'resting' states. The pulsed form reacts aerobically with sulfide to form two intermediates, 'P' and 'F', otherwise involved in the reaction of oxygen with reduced enzyme. Sulfide can directly reduce the oxygen-reactive a3CuB binuclear centre in the pulsed state. The resting enzyme does not undergo such a step, but only a very slow one-electron reduction of the electron-transferring haem a. In final reactivation phases, both the steady-state inhibition of catalysis and the accumulation of P and F states are reversed by slow sulfide oxidation. A model for this complex reaction pattern is presented.

A model for the nitric oxide producing nitrite reductase activity of hemoglobin as a function of oxygen saturation.

The production of nitric oxide by the nitrite reductase activity of hemoglobin has been proposed to play a major role in hypoxic vasodilation. The bimolecular reaction rate constant for nitric oxide formation is a complex function of hemoglobin oxygenation stemming from the intrinsic allosteric character of hemoglobin, resulting in an unsymmetrical inverted U shape profile of activity versus oxygen saturation. We present an analysis of the hemoglobin nitrite reductase activity based on the Monod Wyman Changeux (MWC) allosteric model and derive a set of equations that enabled us to express the rate constant of bimolecular reaction of nitrite with hemoglobin as a function of hemoglobin saturation and use this expression to explore the factors controlling the shape of the nitrite reductase activity versus hemoglobin saturation curve. From the value of the maximum reductase activity, we derive equations to calculate microscopic nitrite reductase reaction rate constants for the R and T quaternary states. We have also developed two methods to parameterize the MWC model based on the Hill equation, with its parameters, h and P50, and the knowledge that these two descriptions of the binding curve coincide in the region of the curve where h is defined. This has allowed the calculation of the hemoglobin nitrite reductase activity rate profiles for the human hemoglobin and for bovine hemoglobin. The properties of these rate profiles are discussed.

Modulating hemoglobin nitrite reductase activity through allostery: a mathematical model.

The production of nitric oxide by hemoglobin (Hb) has been proposed to play a major role in the control of blood flow. Because of the allosteric nature of hemoglobin, the nitrite reductase activity is a complex function of oxygen partial pressure PO2. We have previous developed a model to obtain the micro rate constants for nitrite reduction by R state (kR) and T state (kT) hemoglobin in terms of the experimental maximal macro rate constant kNmax and the corresponding oxygen concentration PO2max. However, because of the intrinsic difficulty in obtaining accurate macro rate constant kN, from available experiments, we have developed an alternative method to determine the micro reaction rate constants (kR and kT) by fitting the simulated macro reaction rate curve (kN versus PO2) to the experimental data. We then use our model to analyze the effect of pH (Bohr Effect) and blood ageing on the nitrite reductase activity, showing that the fall of bisphosphoglycerate (BPG) during red cell storage leads to increase NO production. Our model can have useful predictive and explanatory power. For example, the previously described enhanced nitrite reductase activity of ovine fetal Hb, in comparison to the adult protein, may be understood in terms of a weaker interaction with BPG and an increase in the value of kT from 0.0087M(-1)s(-1) to 0.083M(-1)s(-1).

Modeling hemoglobin nitrite reductase activity as a mechanism of hypoxic vasodilation?

The brain's response to hypoxia is to increase cerebral blood flow (CBF). However, the molecular mechanism underpinning this phenomenon is controversial. We have developed a model to simulate brain blood flow and oxygen metabolism called BRAINSIGNALS. This model is primarily designed to assist in the interpretation of multimodal noninvasive clinical measurements. However, we have recently used this model to test the feasibility of a range of molecular mechanisms proposed to explain hypoxic vasodilation. An increase in the concentration of the vasodilator nitric oxide (NO) at low pO2 is a feature of many such mechanisms. One model suggests that mitochondrial cytochrome c oxidase (CCO) catalyzes the metabolism of NO. This metabolism declines at low pO2, resulting in an increase in the steady-state levels of NO and a consequent increase in CBF. Using BRAINSIGNALS we were able to model this effect. However, the increases in NO and CBF occurred at far lower pO2 values than predicted from physiological data (Rong et al. 2013 Adv Exp Med Biol. 765, 231-238). The aim of the present study was to test an alternative mechanism, one that actively generates NO as pO2 drops, namely, the reduction of nitrite to NO by deoxyhemoglobin. In this mechanism, NO synthesis has a maximum of NO production near the hemoglobin p50. The addition of this mechanism resulted in a significantly better fit to the experimental data of the CBF(PaO2) curve.

Using portable NIRS to compare arm and leg muscle oxygenation during roller skiing in biathletes: a case study.

Portable near-infrared spectroscopy (NIRS) has been shown to be a useful and reliable tool for monitoring muscle oxygenation and blood volume changes during dynamic exercise in elite athletes. The wearable nature of such technology permits the measurement of specific muscles/muscle groups during realistic sport-specific exercise tasks in an outdoor environment. The aim of this case study was to observe the effect on arm and leg muscle oxygenation of roller skiing over a typical outdoor racing course. Such information is required by coaches in order to ascertain whether an athlete is using the correct technique at different stages of the course. Two wearable NIRS devices (PortaMon, Artinis Medical Systems) were used to compare muscle tissue oxygen saturation (TSI%) and total haemoglobin (tHb) changes in the quadriceps muscle group (vastus lateralis) and a muscle of the upper arm (triceps) during roller skiing. During the flat section, quadriceps ΔTSI remained steady in both subjects, whereas triceps ΔTSI showed a reduction (-10 %). During the steep uphill section of the course, arm and leg TSI decreased equally in one subject (ΔTSI = -10 %), whereas there was a difference between the two muscle groups in the other subject (ΔTSIquadriceps = -2 %; ΔTSItriceps = -7 %). A difference was also seen between subjects during the downhill section of the course. This study presents the first example of the use of portable NIRS to assess oxygenation and blood volume changes in multiple muscle groups during roller skiing in a realistic, outdoor setting.

The use of portable NIRS to measure muscle oxygenation and haemodynamics during a repeated sprint running test.

Portable near-infrared spectroscopy (NIRS) devices were originally developed for use in exercise and sports science by Britton Chance in the 1990s (the RunMan and microRunman series). However, only recently with the development of more robust, and wireless systems, has the routine use in elite sport become possible. As with the medical use of NIRS, finding applications of the technology that are relevant to practitioners is the key issue. One option is to use NIRS to track exercise training-induced adaptations in muscle. Portable NIRS devices enable monitoring during the normal 'field' routine uses to assess fitness, such as repeat sprint shuttle tests. Knowledge about the acute physiological responses to these specific tests has practical applications within team sport training prescription, where development of both central and peripheral determinants of high-intensity intermittent exercise needs to be considered. The purpose of this study was to observe NIRS-detected parameters during a repeat sprint test. We used the PortaMon, a two wavelength spatially resolved NIR spectrometer manufactured by Artinis Inc., to assess NIR changes in the gastrocnemius muscle of both the left and right leg during high-intensity running. Six university standard rugby players were assessed (age 20 ± 1.5 years; height 183 ± 1.0 cm; weight 89.4 ± 5.8 kg; body fat 12.2 ± 3.0 %); the subjects completed nine repeated shuttle runs, which incorporated forward, backward and change of direction movements. Individual sprint time, total time to complete test, blood lactate response (BL), heart rate values (HR) and haemoglobin variables (ΔHHb, ΔtHb, ΔHbO2 and ΔTSI%) were measured. Total time to complete the test was 260 ± 20 s, final blood lactate was 14.3 ± 2.8 mM, and maximal HR 182 ± 5 bpm. NIRS variables displayed no differences between right and left legs. During the test, the group-averaged data showed a clear decrease in HbO2 (max. decrease 11.41 ± 4.95 µM), increase in HHb (max. increase 17.65 ± 4.48 µM) and drop in %TSI (max. drop - 24.44 ± 4.63 %). tHb was largely unchanged. However, large interindividual differences were seen for all the NIRS parameters. In conclusion, this observational study suggests that a portable NIRS device is both robust and sensitive enough to detect haemoglobin changes during a high-intensity repeated shuttle run test. It therefore has the possibility to be used to assess exercise training-induced adaptations following a specific training protocol. However, it is at present unclear, given the individual variability, whether NIRS can be used to assess individual performance. We recommend that future studies report individual as well as group data.

NIRS measurements with elite speed skaters: comparison between the ice rink and the laboratory.

Wearable, wireless near-infrared (NIR) spectrometers were used to compare changes in on-ice short-track skating race simulations over 1,500 m with a 3-min cycle ergometry test at constant power output (400 W). The subjects were six male elite short-track speed skaters. Both protocols elicited a rapid desaturation (∆TSI%) in the muscle during early stages (initial 20 s); however, asymmetry between right and left legs was seen in ΔTSI% for the skating protocol, but not for cycling. Individual differences between skaters were present in both protocols. Notably, one individual who showed a relatively small TSI% change (-10.7%, group mean = -26.1%) showed a similarly small change during the cycling protocol (-5.8%, group mean = -14.3%). We conclude that NIRS-detected leg asymmetry is due to the specific demands of short-track speed skating. However, heterogeneity between individuals is not specific to the mode of exercise. Whether this is a result of genuine differences in physiology or a reflection of differences in the optical properties of the leg remains to be determined.

Can mitochondrial cytochrome oxidase mediate hypoxic vasodilation via nitric oxide metabolism?

The brain responds to hypoxia with an increase in cerebral blood flow (CBF). Many mechanisms have been proposed for this hypoxic vasodilation, but none has gained universal acceptance. Although there is some disagreement about the shape of the relationship between arterial oxygen partial pressure (PaO(2)) and CBF, it is generally agreed that CBF does not increase until the PaO(2) reaches a threshold value. We used a previously published computational model of brain oxygen transport and metabolism (BRAINSIGNALS) to test possible molecular mechanisms for such a threshold phenomenon. One suggestion has been that a decrease in the metabolism of nitric oxide by mitochondrial cytochrome c oxidase (CCO) at low PaO(2) could be responsible for raising NO levels and the consequent triggering of the hypoxic blood flow increase. We tested the plausibility of this mechanism using the known rate constants for NO interactions with CCO. We showed that the shape of the CBF-PaO(2) curve could indeed by reproduced, but only if NO production by the enzyme nitric oxide synthase had a very low Michaelis constant K (m) for oxygen. Even then, in the current version of BRAINSIGNALS the NO-induced CBF rise occurs at much lower PaO(2) than is consistent with the in vivo data.

Modelling blood flow and metabolism in the piglet brain during hypoxia-ischaemia: simulating brain energetics.

We have developed a computational model to simulate hypoxia-ischaemia (HI) in the neonatal piglet brain. It has been extended from a previous model by adding the simulation of carotid artery occlusion and including pH changes in the cytoplasm. Here, simulations from the model are compared with near-infrared spectroscopy (NIRS) and phosphorus magnetic resonance spectroscopy (MRS) measurements from two piglets during HI and short-term recovery. One of these piglets showed incomplete recovery after HI, and this is modelled by considering some of the cells to be dead. This is consistent with the results from MRS and the redox state of cytochrome-c-oxidase as measured by NIRS. However, the simulations do not match the NIRS haemoglobin measurements. The model therefore predicts that further physiological changes must also be taking place if the hypothesis of dead cells is correct.

Engineering tyrosine-based electron flow pathways in proteins: the case of aplysia myoglobin.

Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate. Aplysia fasciata myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed.

Nitrite and nitrate reduction by molybdenum centers of the nitrate reductase type: computational predictions on the catalytic mechanism.

Nitrate reductases (NRs) are enzymes that catalyze reduction of nitrate to nitrite using a molybdenum cofactor. In an alternative reaction, plant NRs have also been shown to catalyze reduction of nitrite to nitric oxide, and this appears to be a major source of nitric oxide synthesis in plants, although other pathways have also been shown. Here, density functional theory (DFT) results are shown, indicating that although nitrate is thermodynamically the preferred substrate for the NR active site, both nitrite and nitrate are easily reduced to nitrite and NO, respectively. These mechanisms require a Mo(IV) state. Additionally, in the case of the nitrite, linkage isomerism is at work and controlled by the metal oxidation state, and reduction is, unlike in the nitrate case, dependent on protonation. The data may be relevant to other molybdenum enzymes with similar active sites, such as xanthine oxidase.

Time course of the haemodynamic response to visual stimulation in migraine, measured using near-infrared spectroscopy.

BACKGROUND: In patients with migraine, an abnormally large haemodynamic response to epileptogenic visual stimulation has previously been observed, consistent with the hypothesis of a cortical hyperexcitability. Ophthalmic filters have been used in the treatment of migraine, and they reduce the haemodynamic response. METHODS: The present study used near-infrared spectroscopy (NIRS) to characterise the haemodynamic response to a range of visual stimuli in 20 patients with migraine (15 with aura and 5 without) and paired controls in order to assess the effect of ophthalmic treatment. In an initial study, the response to three stimuli (chequerboard, and two gratings of different spatial frequency) was measured. In a second study, using the mid-spatial frequency grating as stimulus, the response was compared when precision spectral filters (PSF), grey filters or filters of control colour were worn as ophthalmic lenses. RESULTS: In the first study the time course of the response differed between the groups. The difference was most distinct for the grating with mid-spatial frequency. In the second study the PSF broadened (normalised) the haemodynamic response in migraineurs relative to controls, consistent with fMRI BOLD findings and suggesting a physiological mechanism for their reported efficacy. In neither study were there differences in the amplitude of the response between migraine and control groups or indeed between filters. CONCLUSION: The time course of the functional response as measured by NIRS may be an effective tool to track therapy with PSF and explore the mechanisms of visual stress in migraine.