The endogenous vasodilator nitric oxide (NO) is metabolized in tissues in an oxygen-dependent manner. In skeletal and cardiac muscle, high concentrations of myoglobin (Mb) function as a potent NO scavenger. However, the Mb concentration is very low in vascular smooth muscle, where low concentrations of cytoglobin (Cygb) may play a major role in metabolizing NO. Questions remain regarding how low concentrations of Cygb and Mb differ in terms of NO metabolism, and the basis for their different cellular roles and functions. In this study, electrode techniques were used to perform comparative measurements of the kinetics of NO consumption by Mb and Cygb. UV/Vis spectroscopic methods and computer simulations were performed to study the reaction of Mb and Cygb with ascorbate (Asc) and the underlying mechanism. It was observed that the initial rate of Cygb3+ reduction by Asc was 415-fold greater than that of Mb3+. In the low [O2] range (0–50 μm), the Cygb-mediated NO consumption rate is ~ 500 times more sensitive to changes in O2 concentration than that of Mb. The reduction of Cygb3+ by Asc follows a reversible kinetic model, but that of Mb3+ is irreversible. A reaction mechanism for Cygb3+ reduction by Asc is proposed, and the reaction equilibrium constants are determined. Our results suggest that the rapid reduction of Cygb by cellular reductants enables Cygb to efficiently regulate NO metabolism in the vascular wall in an oxygen-dependent manner, but the slow rate of Mb reduction does not show this oxygen dependence.
Recent evidence has shown that cytoglobin (Cygb) is expressed not only in adventitial fibroblasts but also in smooth muscle cells [1, 2], playing a role in vascular nitric oxide (NO) catabolism. NO is an important signaling molecule regulating vascular tone or resistance and controlling blood flow [3, 4]. The rate of NO metabolism in the intervascular tissue  and in the vascular wall  is sensitive to changes in O2 concentration. Cygb metabolizes NO in an O2-dependent manner [1, 7, 8]. We have recently characterized the kinetics of O2-dependent NO metabolism by Cygb in the presence of cellular reductants, and demonstrated that Cygb sensitively transduces a change in O2 concentration to a change in the rate of NO consumption , allowing Cygb to sense and regulate the NO diffusion distance in the vascular wall in response to changes in O2 concentration. However, metabolism of NO is not unique to Cygb. Other globins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin also metabolize NO [7, 8, 10, 11], and the reaction rates of NO with oxyhemoglobin, oxymyoglobin (Mb2+O2), oxyneuroglobin and oxycytoglobin (Cygb2+O2) are very rapid and similar to each other.
Each of the globins has a unique pattern of cellular expression and localization. Hb is mainly located in red blood cells , Mb is mainly located in heart and skeletal muscles , and neuroglobin is mainly present in neurons [14, 15]. Cygb has been found in fibroblasts and in the vascular wall [1, 2, 16]. The concentration of hemes in blood is ~ 8 mm, and the concentration of Mb in heart and skeletal muscles is several hundred micromoles or higher. At such high globin concentrations, the rates of NO consumption in blood, heart and skeletal muscles are very rapid, such that most NO molecules are scavenged by Hb or Mb in these locations [17-21]. Unlike Hb and Mb, the Cygb concentration in vivo is in the low micromolar range [22, 23]. At this low concentration, Cygb does not effectively scavenge NO in the vascular wall, but probably plays a role in regulation of vascular NO concentration. Interestingly, Cygb has been reported to have a binding site for ascorbate that may facilitate its role as an effective reducing substrate . Cygb is considered to have a common evolutionary ancestor with Mb . Mb concentrations in the vascular wall are very low, if present at all . Thus, the preferential expression of Cygb over Mb in the smooth muscle suggests that there is an important functional benefit or role provided by Cygb but not Mb.
Questions remain regarding whether Cygb and Mb have similar behavior in terms of their reaction rate and kinetics of NO metabolism, and the basis for the different roles and functions of these two similar globins. In order to address these questions, we compare Cygb and Mb with regard to their ability to regulate the O2-dependent NO consumption rate and with regard to their rate of reduction by the cellular reductant ascorbate. The reaction mechanism of Cygb reduction by ascorbate is also studied and characterized.
Effect of oxygen concentration on the rate of NO consumption by Mb compared to Cygb
It was previously demonstrated that the rate of NO consumption by Cygb (VCygb-NO) significantly decreases when oxygen concentrations change from 200 μm to 0 μm . In this study, we measured the rate of NO consumption by 0.3 μm Mb in the presence of 300 μm Asc and 400 units/mL superoxide dismutase with varying oxygen concentration (Fig. 1A). The rate of NO consumption by Mb (VMb-NO) only slightly decreases as the O2 concentration decreases. In contrast, NO consumption by 0.3 μm Cygb, 300 μm Asc and 400 units/mL superoxide dismutase is much faster at an oxygen concentration of 200 μm, and the rate of NO consumption significantly decreased when the O2 concentration was changed from 200 μm (room air) to almost 0 μm (anaerobic conditions) (Fig. 1B).
Reduction of Mb3+ by ascorbate
A typical change in the UV/Vis spectrum for reduction of Mb3+ by Asc (10 mm) under anaerobic conditions is shown in Fig. 2A. The Soret band of Mb3+ (409 nm) shifts to that of deoxy Mb2+ (434 nm) gradually with time, indicating Asc-dependent reduction of Mb3+ to Mb2+. The reduction rate of Mb3+ by Asc was reported as second-order, i.e. first-order with respect to both the Mb and Asc concentrations . As the Asc concentrations in the experiments were much greater than the Mb concentration, the reduction of Mb3+ by Asc in our experiments is a pseudo first-order reaction. Using the time course of the change in absorbance at 409 nm, we determined the pseudo first-order rate constant (k′Mb) of Mb3+ reduction at four Asc concentrations (10, 30, 50 and 100 mm). The experiments at each concentration were performed five times. The second-order rate constant of Mb reduction by Asc was then obtained by plotting k′Mb versus Asc concentration as shown in Fig. 2B. The second-order rate constant of Mb reduction by Asc (kMb) was determined as (8.7 ± 0.3) x 10−2·m−1·s−1 (n =5).
Computer simulations of NO consumption by Mb compared to Cygb
Using the rate constants reported in the literature and measured in our experiments (Table 1), we performed computer simulations of VMb-NO and VCygb-NO at varying O2 concentrations (using Eqn (6) for Mb and previously published Eqn  for Cygb). The experimental parameters in the equations, such as Mb or Cygb concentration [E], Asc concentration [A], oxygen concentration [O2] and NO concentration [NO], are the same as the values used in the experiments. As some rate constants and equilibrium constants in Eqn (6) at 37 °C are not available in the literature, the values of these parameters at 37 °C had to be estimated from available data at other temperatures. The reported k1 for Mb ranges from 3.1 x 107m−1·s−1 to 3.7 x 107m−1·s−1 in the temperature range 10–25 °C [10, 27, 28]. Based on this temperature dependence of k1 for Mb, we used a value of k1 = 4.5 x 107m−1·s−1 for Mb at 37 °C. The rate constant k2 for Mb is measured in our experiments as kMb. The reported k4 for Mb at 20 °C is 1.7 x 107m−1·s−1 and the k−4 at 20 °C is 1.2 x 10−4·s−1, so the value of k−4/k4 at 20 °C is 7.1 pm. Given that, when the temperature changes from 20 °C to 37 °C, the k−3/k3 of Mb increases almost fivefold , we estimated the k−4/k4 of Mb at 37 °C as 35 pm, assuming that k−4/k4 and k−3/k3 have a similar temperature dependence. Simulated curves (solid lines) for VCygb-NO and VMb-NO versus [O2] are shown in Fig. 3. The simulated curves are a good fit with the experimental VCygb-NO (closed triangles) and VMb-NO (closed circles) versus [O2]. From the slopes of NO decay curves immediately after each NO concentration peak (Fig. 1), we obtain an NO decay rate that is the sum of NO consumption (Vtotal) by Cygb or Mb, by O2 and by physical diffusion out of the solution. The experimental data for VMb-NO and VCygb-NO in Fig. 3 were obtained by subtracting the rate of NO diffusion out of the solution from the Vtotal for Mb and Cygb, respectively . The final parameters used in the simulated curve are listed in bold in Table 1. Our simulations show that the slope of the VMb-NO–[O2] plot (Fig. 3) is mainly contributed by the NO autoxidation rate Vau rather than the Mb-mediated NO consumption rate VNO. In contrast, VCygb-NO is mainly contributed by the Cygb-mediated NO consumption rate VNO rather than the NO autoxidation rate Vau.
Table 1. Kinetic and equilibrium constants involved in oxygen-dependent NO consumption by Cygb and Mb
The rate equations for Mb and Cygb in this study and our previous paper  were derived under steady-state conditions at any given NO concentration. The in vivo NO concentration at a certain location and a certain time point may be considered as a steady-state concentration achieved by continuous NO generation and NO consumption. The NO concentration used in this study is 500 nm. Although the NO concentrations used in tests of vascular activity may even be micromolar, the physiological NO concentration in the vascular wall for dilating blood vessels may be much lower, which is in the nanomolar range or below . Recently, by using ultrasensitive detector cells, it was observed that the mean NO concentrations in the cerebellum and the hippocampus are 200 pm or less . Direct experimental measurements of Cygb-mediated NO metabolism at the nanomolar and sub-nanomolar NO concentration range in our experiments were not possible because of the detection limit of the NO electrodes. To examine how O2 regulates VCygb-NO under physiological conditions ([NO] in the nanomolar and sub-nanomolar range  and [Asc] in the millimolar range ), we performed computer simulations based on Eqns (6) and (7) and previously published rate equation  to determine changes in VCygb-NO and VMb-NO versus O2 concentration at three NO concentrations: 10 nm, 1 nm and 0.1 nm (Fig. 4). In these simulations, we defined VCygb-NO and VMb-NO at PO2 = 100 torr (13.3 kPa or [O2] = 133 μm at 37 °C in buffer solution) as (V100)Cygb and (V100)Mb, respectively. Note that in experiments in air (21% O2) at 37 °C, PO2 is ~ 20 kPa after correcting for saturated vapor pressure, and the O2 concentration in buffer solution is ~ 200 μm. The conversion factor from PO2 (kPa) to O2 concentration (μm) is 10 μm/kPa, Thus, when PO2 is 13.3 kPa or 1.33 kPa, the corresponding O2 concentration in buffer solution at 37 °C is 133 μm or 13 μm, respectively. The maximal PO2 in arteries under normoxic conditions is almost 13.3 kPa [33, 34], so we consider (V100)Cygb and (V100)Mb to be the maximal rate of NO metabolism that may be achieved at a given NO concentration if the reaction occurs in vivo under normoxic conditions. Both (V100)Cygb and (V100)Mb are dependent on NO concentration. To compare the O2 dependence of VCygb-NO and VMb-NO at various NO concentrations, we normalized the rate of NO metabolism (VCygb-NO and VMb-NO) by dividing VCygb-NO and VMb-NO by (V100)Cygb and (V100)Mb, respectively. The normalized rate of NO metabolism increases as [O2] increases. The O2 concentration range corresponding to the normalized rate of NO metabolism below 0.9 is the main working range for O2 concentration to regulate the rate of NO metabolism. From the simulated curves in Fig. 4, this working range of [O2] for regulating the Cygb-mediated NO metabolism ranges from 0 to 41 μm (4.13 kPa at 37 °C). In contrast, the [O2] working range for regulating Mb-mediated NO metabolism ranges from 0 to 0.43 μm (0.043 kPa), almost 100 times lower than that for Cygb-mediated NO metabolism.
Effect of oxygen concentration on various globin species
The concentrations of each globin species in the reaction system (including 0.5 μm NO, 300 μm Asc, 400 units/mL superoxide dismutase, 0.3 μm Mb or Cygb and varied [O2]) were calculated from Eqns (8)-(11) or from previously published equation . The simulated concentration changes of Cygb3+, Cygb2+NO, Cygb2+O2, Mb3+, Mb2+NO and Mb2+O2 versus [O2] are shown in Fig. 5. The concentrations of Cygb3+, Cygb2+NO and Cygb2+O2 were observed to significantly change with [O2], but the concentrations of Mb3+, Mb2+NO and Mb2+O2 change very little when [O2] is reduced from 200 μm to ~ 0.2 μm (Fig. 5A,B). When [O2] varies from 0 to 50 μm, the Mb2+O2 concentration only increases from 0 to 0.35 pm, but the Cygb2+O2 concentration increases much more from 0 to 0.17 nm (Fig. 5B). Therefore, the concentration increase for Cygb2+O2 is almost 500 times greater than the concentration increase for Mb2+O2 in the low oxygen concentration range between 0 and 50 μm.
Kinetic measurements of Cygb3+ and Mb3+ reduction by ascorbate at various concentrations
As Cygb3+ reduction by Asc is much faster than for Mb3+, we further examined whether the mechanism for Cygb3+ reduction is different from that for Mb3+ reduction. It was observed the reduction of Mb3+ to Mb2+ by either 10 mm or 30 mmAsc went to completion, albeit rather slowly (Fig. 6A). In contrast, Asc only partially reduces Cygb3+ to Cygb2+, with the final ratio of Cygb2+ concentration to the initial Cygb3+ concentration increasing with Asc concentration, reaching an equilibrium between the reactants and products at each given Asc concentration. To simultaneously record Cygb3+ reduction (absorbance peak at 416 nm) and Cygb2+ formation (absorbance peak at 428 nm), repetitive scanning from 350–700 nm was performed using a spectrophotometer while injecting various concentrations of Asc (0.1, 0.2, 0.7, 2, 7 and 20 mm) into the chamber. From these recorded spectra, we determined the kinetic characteristics of conversion of Cygb3+ to Cygb2+ by plotting the absorbance changes versus time at the wavelengths 416 and 428 nm (Fig. 6B).
Reaction mechanism of Cygb3+ reduction by Asc
The large difference between reduction of Mb3+ and Cygb3+ by Asc suggests that the reaction kinetics and mechanism for Cygb3+ reduction by Asc differ from Mb3+ reduction by Asc. The formation of different equilibrium concentrations between Cygb2+ and Cygb3+ at different concentrations of Asc suggests that the reduction of Cygb3+ by Asc is a reversible reaction. Furthermore, 10 μm Cygb3+ cannot be completely reduced even by addition of as much as 30 mm Asc. Based on these observations, we propose the following reaction mechanism for Cygb3+ reduction by Asc:
Assuming that the initial Cygb3+ concentration is c0, the concentration of intermediate Cygb3+A is x, the concentration of product Cygb2+A+ is y, and the Asc concentration [A] is a constant in each measurement because the Asc concentration greatly exceeds c0, we obtain the following equations when equilibrium is reached:
where K1 = k1/k−1. Substitution of Eqn (4) into Eqn (3) gives:
Eqn (5) indicates that the ratio of the equilibrium concentration of Cygb2+ (y) to the initial Cygb3+ concentration (c0) increases with Asc concentration but does not approach 1 unless K2 tends to infinity. We further experimentally examined formation of Cygb2+ in the presence of various Asc concentrations under anaerobic conditions at 37 °C by recording the change in absorbance at 428 nm over time using a spectrophotometer. Using the Cygb2+ equilibrium concentration (y) at each Asc concentration ([A]), we plot the ratio y/c0 (or Cygb2+/[Cygb3+]0) versus [A], closed circles) and then use Eqn (5) to fit the experimental data. As shown in Fig. 7, the best-fitted curve (solid line) is in excellent agreement with the experimental data. From the best-fitting curve, we obtained a value of K1 K2 of 0.0633 ± 0.002 mm−1 and a value of K1 (1 + K2) of 0.0751 ± 0.0035 mm−1 (n =7). The values of K1 and K2 calculated from the measured K1 K2 and K1 (1 + K2) are 0.012 ± 0.006 mm−1 and 5.4 ± 1.8, respectively.
The rate of Mb-mediated NO consumption (Fig. 1A) is much less regulated by oxygen concentration than that of Cygb-mediated NO consumption (Fig. 1B). This large difference implies that the rate of Cygb3+ reduction by Asc may be much greater than the rate of Mb3+ reduction by Asc. The rate of Mb3+ reduction by Asc is relatively slow (Fig. 2A) and the apparent first-order rate constant is linear with Asc in the concentration range we examined (Fig. 2B). In contrast, the rate of Cygb reduction by Asc is much faster and the rate is not linear with Asc concentration . Comparison of k2 for Cygb with k2 for Mb (Table 1) shows that the reduction of Cygb3+ by Asc is 415 times faster than the reduction of Mb3+ by Asc. Using Eqn (7), the rate equation for Cygb , and the kinetic parameters in Table 1, we calculated VMb-NO/[O2] and VCygb-NO/[O2] curves, which fit the experimental data very well (Fig. 3). Our results show that, in the presence of hundred micromolar concentrations of Asc and sub-micromolar concentrations of globin, Mb-mediated NO consumption is much slower than the NO autoxidation rate, making autoxidation the main component in the VMb-NO term for the reaction with Mb. In contrast, Cygb-mediated NO consumption rather than NO autoxidation is the main component in the VCygb-NO term. In Fig. 4, we demonstrate the O2 dependence of Cygb- and Mb-mediated NO metabolism at three NO concentrations (10, 1 and 0.1 nm), showing that the major working range of PO2 for Cygb-mediated NO metabolism in our experiments is within 0–4.13 kPa (41 μm at 37 °C). The curve shifts to the left when [NO] decreases from 10 to 1 nm, but the curve shift is very small when [NO] is further decreased from 1 to 0.1 nm. As a result of this shift, O2 more efficiently regulates the Cygb-mediated NO consumption within the physiological range of NO concentration (low nanomolar concentrations or below) when PO2 is lower than 4.13 kPa. It was reported that, for wild-type rats under normoxic conditions (21% O2 in air), the O2 partial pressure (PO2) is ~ 13.3 kPa in the arteries and 27 torr (3.6 kPa) in the tissues . When the inspired O2 tension decreases from 21% to 7% (hypoxia), the PO2 in tissues decreases below 10 torr (1.33 kPa) . Thus PO2 in these hypoxic tissues is within the major working range of PO2 for Cygb-mediated NO metabolism at physiological NO concentrations. Under the same conditions, the [O2] working range for Mb-mediated NO metabolism is only 0.32 torr (0.043 kPa), indicating that the rate of NO metabolism by Mb in the vascular wall does not change with [O2] until [O2] is below 0.043 kPa. However, even if the Mb-mediated NO consumption is regulated by O2 concentration when [O2] drops below 0.043 kPa, this O2 dependence of Mb-mediated NO metabolism is not important in the vascular wall because it is smaller than the rate of NO autoxidation (Fig. 3).
Computer simulations show that changes in oxygen concentration have little effect on concentrations of Mb3+, Mb2+NO and Mb2+O2 under the experimental conditions of this study, and the majority of Mb exists in the form of Mb3+ due to the slow rate of Mb3+ reduction by Asc (Fig. 5). In contrast, concentrations of Cygb3+, Cygb2+NO and Cygb2+O2 are very sensitive to changes in oxygen concentration. The ability of Cygb to efficiently transduce signals from changes in [O2] into changes in the rate of NO consumption may be attributed to the significant formation rate of Cygb2+O2. As the concentration increase of Cygb2+O2 is almost 500 times greater than the concentration increase of Mb2+O2 in the oxygen concentration range between 0 and 50 μm, and the rate of NO dioxygenation by Cygb and Mb is proportional to the Cygb2+O2 and Mb2+O2 concentration, respectively, this indicates that Cygb is almost 500 times more sensitive than Mb at ‘detecting’ changes of oxygen concentration to regulate the rate of NO consumption in the presence of ascorbate as reductant.
It is interesting that the reaction kinetics for the reduction of Cygb by Asc are very different from those for reduction of Mb by Asc (Fig. 6). The existence of a corresponding equilibrium concentration of Cygb2+ product for a given Asc concentration indicates that the reduction of Cygb3+ by Asc is reversible. To explain the fact that Cygb3+ cannot be completely reduced by Asc even with Asc in gross excess, we suggest, as reported previously , that Asc binds to Cygb3+ prior to the reduction step. Thus, as the reduction is reversible, Cygb3+A cannot be completely converted into Cygb2+A+. Eqn (5) derived from the reaction mechanism shown in Eqn (1) fits the experimental data very well (Fig. 7), indicating that the proposed reaction mechanism explains the experimental results for the reduction of Cygb3+ by Asc. The physiological role of the reversible reduction of Cygb remains to be further understood; however, existence of this reversible reduction may prevent Cygb3+ from being fully reduced to Cygb2+ in hypoxia or anoxia. This lower concentration of reduced Cygb reduces the production of reactive oxygen species and decreasing the rate of NO consumption during reoxygenation.
If a globin is to act as an oxygen sensor for the regulation of free NO and secondary vasodilation in the vascular wall, the rate of NO catabolism by this globin must be appropriate. If the NO decay rate is too high in the vascular wall, the effective NO concentration may be too low to regulate vascular tone. Conversely, if the globin-mediated rate of NO catabolism is too low, this globin cannot be the major catabolic pathway to control NO concentration. Cygb appears to meet these requirements. The Cygb concentration in tissue is almost four orders of magnitude lower than the Hb concentration in blood and two to three orders of magnitude less than the Mb concentration in skeletal and cardiac muscle. In this low concentration range, Cygb cannot act as an efficient NO scavenger as Hb and Mb do in red blood cells [19, 21], skeletal and cardiac muscle [18, 20], but it is able to efficiently regulate the rate of NO metabolism in the vascular wall because Cygb3+ may be rapidly recycled to Cygb2+ by cellular reductants to continuously metabolize NO. As the rate of Mb reduction by Asc is two orders of magnitude slower than that for Cygb, and the vascular wall contains only very low levels of Mb, vascular Mb cannot efficiently use cellular Asc as a reductant to metabolize NO. However, results have shown that Mb in the myocardium efficiently generates NO by reducing nitrite as O2 concentration decreases . This Mb-mediated NO generation from nitrite has protective effects on ischemia–reperfusion injury. It has been demonstrated that some other reductants and cellular reductases efficiently supply electrons to Cygb3+ for Cygb-mediated NO consumption, but these electron donors are not effective in supporting Mb-mediated NO consumption . Cygb is a bis-histidyl hexacoordinate globin. It has been shown that bis-histidyl hexacoordination in globins facilitates heme reduction by small molecules . These lines of evidence indicate that Cygb is much more efficient than Mb in regulating NO metabolism in the vascular wall, where globin concentrations are in the low micromolar range or less, by using cellular reductants as electron donors. In comparison, Mb is mainly present in skeletal or cardiac muscle, where its high concentration enables O2 storage, in turn facilitating O2 diffusion in cardiac and skeletal muscles. The high concentration of Mb in these tissues, together with its high affinity for O2, plays a role in providing oxygen to the tissue under severe hypoxia. Its high rate of NO dioxygenation plays an additional role in the oxidative degradation of NO in these muscles.
The rate of metCygb reduction by Asc is hundreds of times greater than the rate of metMb reduction by Asc. As a result, Cygb regulates the rate of NO catabolism in response to a change in oxygen concentration almost 500 times more efficiently than its family member Mb. In addition to the large difference in the rate constants between Cygb reduction and Mb reduction by Asc, the reduction of Cygb by Asc is reversible, such that Cygb3+ cannot be fully reduced to Cygb2+ in the presence of millimolar or even tens of millimolar concentrations of Asc. The rapid reduction of Cygb by cellular reductants enables Cygb to efficiently regulate oxygen-dependent NO metabolism in the vascular wall.
Expression and purification of recombinant Cygb
The expression plasmid for Cygb (human Cygb cDNA in pET3ac, Novagen, Merck KGaA, Darmstadt, Germany) was obtained from Thorsten Burmester (Institute of Zoology and Zoological Museum, University of Hamburg, Germany) and transformed into Escherichia coli strain C41 (DE3)pLysS. Cells were grown, harvested and lysed as described previously . A 35% ammonium sulfate precipitation was performed on the supernatant, the pellet was discarded, and the supernatant was dialyzed against 2 L of 50 mm Tris/HCl, 1 mm dithiothreitol and 0.1 mm EDTA, pH 7.5, with a total of three buffer exchanges. After dialysis, insoluble material was removed by centrifugation (45 000 g for 1 h), and the human Cygb was concentrated to 50 mL using Amicon Ultra-15 centrifugal filters (Millipore) with a 10 000 molecular weight cut-off. Final purification was performed using a GE Healthcare ÄKTA Purifier system with a 50 mL Superloop (GE Healthcare, Piscataway, NJ, USA) for sample loading. A HiPrep 16/10 DEAE FF anion-exchange column (GE Healthcare) was run with a sodium chloride gradient elution, followed by a HiPrep 26/60 Sephacryl S-300 high-resolution size-exclusion column (GE Healthcare) eluted with 50 mm Tris/HCl, pH 7.5, 100 mm NaCl and 0.1 mm EDTA. The protein was concentrated and stored in 50 μL aliquots at −80 °C.
Reduction of Cygb3+ or Mb3+ by ascorbate
The reduction of Cygb3+ and Mb3+ by Asc in a chamber (cuvette) was performed in a similar manner to that described previously . Briefly, the chamber was covered by Parafilm membrane (a semi-transparent, flexible, thermoplastic, and highly waterproof sheet material), and the buffer solution (pH 7.0, 37 °C) in the chamber was de-aerated by bubbling argon gas into the solution for at least 15 min through a tube. After adding 10 μm de-aerated Cygb3+ or Mb3+ into the chamber, the tube was moved to the gas phase above the solution surface, and the argon gas flow was maintained in the chamber during the experiments. After 5–10 min, varying concentrations of de-aerated Asc were then added into the chamber to start the reduction using a Hamilton gas-tight syringe (Hamilton Company, Reno, NV, USA). The reduction of Cygb3+ or Mb3+ in the solution was monitored using a Cary 50 UV/Vis spectrophotometer (Varian Inc. Palo Alto, CA, USA) scanning from 350–700 nm or using a wavelength fixed at the absorbance peak of the corresponding Soret band.
Simultaneous measurements of [NO] and [O2]
The measurements of [NO] and [O2] were performed in a four-port water-jacketed electrochemical chamber (NOCHM-4 from World Precision Instruments, Sarasota, FL) containing 1 or 2 mL HyClone Dulbecco's phosphate-buffered saline (pH 7.0, Thermo Scientific, Chelmsford, MA, USA). An NO electrode and an O2 electrode were connected to an Apollo 4000 electrochemical instrument (WPI). NO solution was prepared as described previously [5, 37]. To measure the rate of NO consumption by Cygb at various oxygen concentrations, and to compare the results with those for Mb, NO (final concentration 0.5 μm) was added to the solution in the presence of 0.3 μm Cygb (or Mb), 300 μm Asc and 400 units/mL superoxide dismutase to measure the rate of NO consumption in room air. Argon gas was then introduced in the chamber headspace to remove O2 from the solution. While the oxygen concentration was gradually decreased by the flow of argon gas, 0.5 μm NO was repeatedly injected into the solution. From the recorded [NO]/t and [O2]/t curves, the oxygen concentration and the NO decay rate (VNO) at each NO peak were measured and used to plot the VNO/[O2] curve.
Computer simulations of NO consumption by Cygb and Mb
Computer simulations of the rate of NO consumption by Cygb and Mb were performed on a PC using matlab software (MathWorks, Natick, MA, USA). The equations used in the simulations for Cygb have been published previously , and the equations for Mb are similar to those for Cygb except that the term kh/k−h is not involved:
In the above equations, VNO is the rate of NO consumption by Mb, [E] is the total Mb concentration, [R] is the Asc concentration, and VMb-NO is the sum of VNO and the rate of NO autoxidation in solution as defined in Eqn (7).
This work was supported by US National Institutes of Health grants HL063744, HL065608 and HL38324. We thank undergraduate students Kaitlyn Boggs, Caty P. Escobar, Rachael Huskey, Yeram Kang and Mariel McGuiness for their volunteer work in our laboratory.