Oxygen binding and nitric oxide dioxygenase activity of cytoglobin are altered to different extents by cysteine modification

Abstract

Cytoglobin (Cygb), like other members of the globin family, is a nitric oxide (NO) dioxygenase, metabolizing NO in an oxygen (O₂)‐dependent manner. We examined the effect of modification of cysteine sulfhydryl groups of Cygb on its O₂ binding and NO dioxygenase activity. The two cysteine sulfhydryls of Cygb were modified to form either an intramolecular disulfide bond (Cygb_SS), thioether bonds to N‐ethylmaleimide (NEM; Cygb_SC), or were maintained as free SH groups (Cygb_SH). It was observed that the NO dioxygenase activity of Cygb only slightly changed (~ 25%) while the P₅₀ of O₂ binding to Cygb changed over four‐fold with these modifications. Our results suggest that it is possible to separately regulate one Cygb function (such as O₂ binding) without largely affecting the other Cygb functions (such as its NO dioxygenase activity).

Abbreviations

Abs_b

the absorbance at baseline or when time t approaches to infinity

Asc

ascorbate

b5

cytochrome b5

b5R

cytochrome b5 reductase

Cygb‐SC

cytoglobin with thioether bonds between a cysteine residue and N‐ethylmaleimide

Cygb‐SH

cytoglobin with free sulfhydryl group

Cygb‐SS

cytoglobin with intramolecular disulfide bond

DT

dithionite

DTT

dithiothreitol

Cytoglobin (Cygb), a member of the globin family, was discovered 15 years ago 1, 2, 3. Like other family members such as hemoglobin (Hb) and myoglobin (Mb), Cygb is a nitric oxide (NO) dioxygenase, which metabolizes NO in an oxygen (O₂)‐dependent manner 4, 5. Cygb is present in splanchnic fibroblasts of various organs 6, fibroblast‐related cell lineages 7, and other cell types such as neurons 8, 9, hepatocytes 10, 11, and vascular smooth muscle cells 12. In vivo, Cygb may play several different roles including tumor‐suppression 13, lipid signaling of oxidative stress 14, ROS protection 15, 16, 17, NO metabolism 4, 12, nitrite reduction18, and regulation of vascular NO concentration 5, 19.

The Cygb monomer contains two exposed cysteine residues (Cys 38 and Cys 83) that enable Cygb to form an intramolecular disulfide bond 20, 21, 22. The formation of this intramolecular disulfide bond greatly increases the dissociation rate constant of a bound intrinsic histidine, resulting in a greater apparent binding constant of extrinsic ligands 21, 22. As a NO dioxygenase, O₂ must bind to Cygb before it metabolizes NO. Under hypoxic conditions, Cygb with intramolecular disulfide bond (Cygb‐SS) holds more O₂ in the form of Cygb(Fe²⁺O₂) than Cygb with free sulfhydryl group (Cygb‐SH) and Cygb with thioether bonds between a cysteine residue and N‐ethylmaleimide (NEM; Cygb‐SC). However, the NO dioxygenase activity of Cygb is limited and effectively controlled by the rate of Cygb reduction 23. Thus, if the rate of Cygb reduction is not altered by modification of the sulfhydryl groups in the Cygb, the NO dioxygenase activity of Cygb may not be greatly affected even under hypoxic conditions.

It was suggested that Cygb in vivo is monomeric 20, 24. However, Cygb dimer and other oligomers have been reported to exist in Cygb preparations 22, 25. Although both Cygb monomer and oligomers are of interest to study, the monomer is reported to be the predominant form both in vivo and in vitro 18, 20, 24. Therefore, in this study, we focus on examination of the effect of modifying the sulfhydryl groups of the cysteine residues in monomeric Cygb on the affinity of O₂ binding and the rate of Cygb‐mediated O₂‐dependent NO metabolism in the presence of cellular reductants.

Materials and methods

Modification of the sulfhydryl groups of the cysteine residues (RSH) of Cygb

The expression and purification process of Cygb was described in our prior reports 5. As isolated Cygb was first treated with dithiothreitol (DTT, C₄H₁₀O₂S₂) in phosphate‐buffered saline (PBS) buffer (pH = 7.4 with 0.1 mm EDTA) on ice for about 30 min to reduce any remaining disulfide bonds in the Cygb preparation, forming a nearly homogeneous solution of Cygb in the sulfhydryl form (Cygb‐SH). This protein was run through a G‐25 column to remove the excess DTT then aliquoted for treatment to modify the free thiol groups as follows: reaction with 2 mm NEM (C₆H₇NO₂ MW: 125.13) to alkylate reactive thiol groups (Cygb‐SC), or 4 mm 4,4′‐dithiodipyridine (4‐PDS, C₁₀H₈N₂S₂ MW: 220.3) to drive disulfide bond formation (Cygb‐SS) at room temperature for 1 h. After the incubation period, the samples were concentrated and again run through a G‐25 column to remove excess reactants. The purity of the three products Cygb‐SS, Cygb‐SH, and Cygb‐SC was determined by comparing the concentration of a given sample of Cygb (measured by the pyridine hemochromagen assay 26) with the concentration of free thiols of the same Cygb sample (measured by the 4‐PDS method 27). The final average percentage of thiol modification is > 90%.

PAGE and Coomassie staining of pure Cygb

Pure Cygb (10 ng) was loaded to 4–20% gradient gels under nonreducing conditions. Protein samples were separated at 150 V for ~ 90 min. At the end of separation, gels were incubated with 1% Coomassie stain in 40% methanol and 10% acetic acid. After 2 h of staining, gels were destained with repeated 15 min incubations with ~ 20 mL of destaining solution (40% methanol and 10% acetic acid) until bands were clear. Gels were then imaged with a Bio‐Rad (Hercules, CA, USA) Versadoc imaging system using quantityone imaging software.

Measurements of O₂ binding and redox state of Cygb(Fe²⁺)

A Cary 50 UV/Vis spectrophotometer was used to measure the changes in absorbance at 428 nm (A₄₂₈; peak absorbance of Cygb(Fe²⁺)) with time in the process of reduction of Cygb(Fe³⁺) and O₂ binding to Cygb(Fe²⁺). Simultaneously, a Clark O₂ electrode was placed in the solution to monitor the changes in O₂ concentration ([O₂], 37 °C). Both A₄₂₈ and [O₂] were sampled at the same rate (10 readings per second). After 1.5 mL of buffer solution and Cygb(Fe³⁺) were added into the cuvette, the cuvette was covered with a Parafilm membrane. An argon gas tube with an outer diameter of 1 mm was inserted into the cuvette that was covered by a parafilm membrane and positioned above the solution surface to keep an argon flow in the cuvette to gradually remove O₂ from the solution. When O₂ in the solution decreased by about half, ~ 1 mm dithionite (DT) was injected into the solution to rapidly scavenge all O₂ and reduce Cygb(Fe³⁺) to Cygb(Fe²⁺) in the cuvette. Although the recordings of O₂ concentration and the absorbance of Cygb(Fe²⁺) were started before injection of DT, time 0 was assigned to the time point at which DT was added into the solution. At this moment, both [O₂] and A₄₂₈ suddenly changed. This time point was used as the starting point for synchronizing the readings of A₄₂₈ and [O₂], which is important for matching the percent of O₂ bound to Cygb(Fe²⁺) with the corresponding [O₂] in the solution. After Cygb was reduced and O₂ was scavenged from both the solution and the gas phase, the gas tube was taken out of the cuvette, and the hole in the Parafilm membrane for the gas tube was sealed by a piece of Parafilm. A syringe needle was used to make a tiny hole on the Parafilm so that O₂ in the air could enter the gas phase of the cuvette slowly. The entered O₂ was gradually dissolved into the test solution to raise the O₂ concentration in the solution at a rate of ~ 0.04–0.06 μm·s⁻¹ to maintain the O₂ binding process near the equilibrium. The dissolved O₂ can bind on Cygb(Fe²⁺) to form Cygb(Fe²⁺O₂).

Electrochemical measurements of the rate of O₂‐dependent NO metabolism by Cygb

The measurements were performed in a four‐port water‐jacketed electrochemical chamber (NOCHM‐4 from WPI, Sarasota, FL, USA) containing 2 mL of Dulbecco's PBS (Thermo Scientific, South Logan, UT, USA) as previously described 5, 19. The NO solution was also prepared as previously described 28, 29. After the NO and O₂ electrodes had stabilized, NO (0.5 μm) was injected into the aerated buffer solution (under room air) in the absence of Cygb and reductants. When the NO concentration decreased to baseline, 0.4 μm Cygb, 400 U·mL⁻¹ superoxide dismutase (SOD) from horseradish (Sigma‐Aldrich, St. Louis, MO, USA) or 50 μm SOD mimetic (GC4419, Galera Therapeutics Inc., Malvern, PA, USA), and either ascorbate (Asc; Sigma‐Aldrich) or cytochrome b5 reductase (b5R) with NADH (Sigma‐Aldrich) and cytochrome b5 (b5) were added to the chamber. Then 0.5 μm NO was injected into the solution to measure the rate of NO consumption under room air. To measure the rate of NO consumption by Cygb at different O₂ concentrations below 200 μm, argon gas tube was introduced into the chamber above the solution through a hole in the cap for removing O₂ from the solution. While [O₂] in the solution was gradually decreased due to the flow of argon gas, equal amounts of 0.5 μm NO were repeatedly injected into the solution. Changes in [O₂] and [NO] over time were recorded by the O₂ and NO electrodes, respectively. From the recorded temporal [NO] and [O₂] curves, the rate of NO decay (V_NO) at each NO peak and the corresponding [O₂] were measured and used to plot the V_NO vs. [O₂] curves.

Spectrophotometric measurements of Cygb(Fe³⁺) reduction by Asc or by the b5R/b5/NADH enzyme‐reducing system

Measurements were performed on a Cary 50 UV/Vis spectrophotometer. After 1.5 mL of buffer solution was added into the cuvette, the cuvette was covered with a Parafilm membrane. A Clark O₂ electrode was placed in the solution to monitor O₂ concentration. The measured O₂ concentrations were recorded by an Apollo 4000 Free Radical Analyzer (WPI) using a Clark electrode. The solution was stirred using a magnetic stirring bar that was placed on the bottom of the cuvette. An argon gas tube was inserted in the cuvette to bubble argon into the solution for 15 min to quickly remove O₂. Before injecting Cygb (for reduction by Asc) or Cygb + b5 + NADH (for reduction by the reducing system b5R/b5/NADH) into the solution, the argon gas tube was placed just above the solution surface to keep an argon flow in the cuvette. About 20 min after injection of Cygb or Cygb/b5/NADH, Asc or b5R was added to the solution to initiate reduction of Cygb(Fe³⁺), respectively. The reaction process was monitored by measuring the changes in absorbance at 416 nm (peak absorbance of Cygb(Fe³⁺)) with time 30.

Equations for determining rate constants of Cygb reduction

The reduction scheme of Cygb(Fe³⁺) reduction by Asc has been proposed in our previous paper 30:

$$A+B\underset{k_1}{\overset{k_1}{\rightleftharpoons }}C\underset{k_2}{\overset{k_2}{\rightleftharpoons }}D$$ (1)

where A is reductant, B is Cygb(Fe³⁺), C is the complex Cygb(Fe³⁺ A), and D is Cygb(Fe²⁺ A ⁺). In the following derivation process, concentrations of A, B, C, D and the total Cygb protein E are presented by [A], [B], [C], [D], and [E], respectively. [E] = [B] + [C] + [D] for any time t. Since E is the total Cygb concentration including B, C, and D, E is considered as a constant in the experiments assuming that Cygb only has the three forms B, C, and D. Concentrations of A, B, C, and D in the beginning of the reaction (t = 0) are written as [A]₀, [B]₀, [C]₀, [D]₀, respectively. At t = 0, we assume that [B]₀ = [E], [C]₀ = [D]₀ = 0. Using steady‐state approximation, the complex C is an intermediate and its concentration is assumed in the steady‐state or d[C]/dt = 0. Thus, we can obtain:

$$\begin{array}{cc}\hfill \frac{d[B]}{dt}& =-k_1[A][B]+k_{-1}[C],\hfill \\ \hfill \frac{d[C]}{dt}& =k_1[A][B]+k_{-2}[D]-(k_{-1}+k_2)[C]=0,\hfill \\ \hfill [B]+[C]+[D]& =[E]\text{and}\frac{d[B]}{dt}=-\frac{d[D]}{dt}\hfill \end{array}$$

Under the steady‐state approximation (dC/dt = 0), the rate of B consumption is equal to the rate of D formation. From the above equations we can obtain (see Data S1):

$$\frac{d[B]}{dt}=\frac{k_{-1}{k}_{-2}[E]}{k_{-1}+k_2+k_{-2}}-\left(\frac{k_{-1}{k}_{-2}+k_1(k_2+k_{-2})[A]}{k_{-1}+k_2+k_{-2}}\right)[B]=-g_1([B]-[B_{\mathrm{b}}])$$ (2)

where g ₁ can be considered as the rate constant of reduction of B by A. In Eqn (3), d[B]/dt approaches 0 as t approaches to infinity. Thus, [B] approaches [B _b] as t approaches infinity. Here, [B _b] is concentration of B at t = ∞. From Eqn (3) we obtain:

$$\text{ln}([B]-[B_{\mathrm{b}}])=-g_{1}t+g_0$$ (3)

where g ₀ is an integration constant. In experiments for measuring the reduction of Cygb(Fe³⁺), we used a UV/Vis spectrophotometer to monitor the changes in absorbance (Abs) at wavelength 416 nm. According to the Beer–Lambert Law, we have:

$$\text{Abs}={\mathrm{\epsilon }}_B[B]+{\mathrm{\epsilon }}_C[C]+{\mathrm{\epsilon }}_D[D]$$ (4)

where ε_B, ε_C and ε_D are the molar extinction coefficients of B, C and D, respectively. Abs is the absorbance at time t. If time t is large (theoretically t approaches infinity), Eqn (4) can be written in the following form:

$$\text{Abs}{}_{\mathrm{b}}={\mathrm{\epsilon }}_B[B_{\mathrm{b}}]+{\mathrm{\epsilon }}_C[C_{\mathrm{b}}]+{\mathrm{\epsilon }}_D[D_{\mathrm{b}}]$$ (5)

where Abs_b is the aborbance as t approaches infinity.

We also have: [D] = [E] − [B] − [C] and [D _b] = [E] − [B _b] − [C _b]. Here, [C _b] and [D _b] are concentrations of C and D as t approaches infinity. Combining these two equations with Eqs (4) and (5) gives:

$$\text{Abs}=({\mathrm{\epsilon }}_B-{\mathrm{\epsilon }}_D)[B]+({\mathrm{\epsilon }}_C-{\mathrm{\epsilon }}_D)[C]+{\mathrm{\epsilon }}_D[E]$$ (6)

$$\text{Abs}{}_{\mathrm{b}}=({\mathrm{\epsilon }}_B-{\mathrm{\epsilon }}_D)[B_{\mathrm{b}}]+({\mathrm{\epsilon }}_C-{\mathrm{\epsilon }}_D)[C_{\mathrm{b}}]+{\mathrm{\epsilon }}_D[E_{\mathrm{b}}]$$ (7)

Then we can get:

$$\text{Abs}-{\text{Abs}}_{\mathrm{b}}=({\mathrm{\epsilon }}_B-{\mathrm{\epsilon }}_D)([B]-[B_{\mathrm{b}}])+({\mathrm{\epsilon }}_C-{\mathrm{\epsilon }}_D)([C]-[C_{\mathrm{b}}])$$ (8)

Under steady‐state approximation, C is a constant during the measurements of absorbance. Thus, we can obtain:

$$[B]-[B_{\mathrm{b}}]=\frac{\text{Abs}-{\text{Abs}}_{\mathrm{b}}}{{\mathrm{\epsilon }}_B-{\mathrm{\epsilon }}_D}$$ (9)

Substituting Eqn (9) into Eqn (3), we have:

$$\text{ln}(\text{Abs}-{\text{Abs}}_{\mathrm{b}})=-g_{1}t+g_0+\text{ln}({\mathrm{\epsilon }}_B-{\mathrm{\epsilon }}_D)=-g_{1}t+g$$ (10)

Equation (10) indicates that the plot of ln(Abs − Abs_b) vs. t from experimental data will generate a straight line with a slope of −g ₁.

Results

PAGE and Coomassie staining of Cygb

PAGE followed by Coomassie staining was performed to characterize each of the Cygb preparations including the reduced protein Cygb‐SH (SH), the oxidized disulfide Cygb‐SS (SS), and NEM‐modified Cygb‐SC (SC). The resolved gel of the three types of Cygb preparations is shown in Fig. 1. The pure Cygb bands appear with a molecular weight of ~ 21 kDa. The bands in the right column are protein markers with molecular weights indicated. In all three of the Cygb preparations, only monomeric Cygb is present. Any possible dimer form may have been converted into monomer in the preparation of Cygb samples with DTT.

Figure 1.

SDS/PAGE analysis of the Cygb preparations (SH, SS and SN represent Cygb‐SH, Cygb‐SS, and Cygb‐SC). Cygb (10 ng) was loaded on PAGE gels under nonreducing conditions. Gels were Coomassie‐stained and imaged. A single band is seen at ~ 21 kDa indicating that only monomeric Cygb was present.

O₂‐binding curves of modified Cygbs

To examine the effect of modifying the sulfhydryl groups of cysteine on the affinity of O₂ binding to Cygbs, we measured O₂‐binding curves for each of the three Cygb preparations at 37 °C. Figure 2 shows typical experimental O₂‐binding curves. It can be seen that the P₅₀ of Cygb has the following order: Cygb‐SS < Cygb‐SH < Cygb‐SC. The mean and standard errors of the P₅₀ values (n = 3) for the three Cygbs are listed in Table 1 (unit in mmHg or Torr).

Figure 2.

Experimental curves of O₂ binding to 3 μm Cygb‐SS, Cygb‐SH, and Cygb‐SC.

Table 1.

Measured P₅₀ (mmHg) for O₂ binding to Cygb

Proteins	Cygb‐SS	Cygb‐SH	Cygb‐SC
Mean	0.7	1.4	2.9
SE	0.1	0.1	0.2

Reduction of modified Cygbs

During NO dioxygenation, O₂ binds to Cygb(Fe²⁺) to form Cygb(Fe²⁺O₂) which can be oxidized to Cygb(Fe³⁺) by NO. To maintain continuity of NO dioxygenation, a reductant (such as Asc) is required for reducing Cygb(Fe³⁺) back to Cygb(Fe²⁺). The reduction of the three Cygb(Fe³⁺) preparations by Asc (1–20 mm) and by b5R (5–120 nm)/b5 (0.5 μm)/NADH (100 μm) was measured with a spectrophotometric assay. It was observed that either Asc or b5R in the presence of b5 (0.5 μm) and NADH (100 μm) cannot fully reduce each of the Cygbs including Cygb‐SH, Cygb‐SC, and Cygb‐SS in the concentration range listed above, but full reduction could be accomplished by 1 mm DT. In Fig. 3 we demonstrated the typical experimental curves for reduction of Cygb‐SH by Asc (Fig. 3A) and b5R/b5/NADH (Fig. 3B). From the experimental data, we plotted ln(Abs − Abs_b) vs. time t based on Eqn (10) (Fig. 3C,D). All plots with Asc as the reductant are linear with time after 2–5 s from the initial injection of Asc (Fig. 3C). From the slopes of these lines, we obtained the pseudo first‐order rate constant g ₁, which (shown as k _Asc or k _b5R in Fig. 3E–F) is linear with reductant concentration [Asc] or [b5R]. In Fig 4A,B, we demonstrated the plots of ln(Abs − Abs_b) vs. time t for Cygb‐SH, Cygb‐SS, and Cygb‐SC reduction by 10 mm Asc or 30 nm b5R in the presence of 0.5 μm b5 and 100 μm NADH. All plots are nearly linear. From the slopes of the lines, we can determine the pseudo first‐order rate constants of reduction of the three Cygbs by Asc (10 mm) or by b5R (30 nm) in the presence of 0.5 μm b5 and 100 μm NADH. The pseudo first‐order rate constants of Cygb‐SH, Cygb‐SS, and Cygb‐SC at other Asc and b5R concentrations are shown in Fig 4C (for Asc) and Fig. 4D (for b5R).

Figure 3.

Reduction of Cygb‐SH by cellular reductants. Measurements of absorbance at 416 nm while 2 μm Cygb‐SH was reduced by different concentrations of Asc (A) or by different concentrations of b5R in the presence of 0.5 μm b5 and 100 μm NADH (B). Plot of ln(Abs − Abs_b) vs. time as 2 μm Cygb‐SH was reduced by varied concentrations of Asc (C), and by varied concentrations of b5R in the presence of 0.5 μm b5 and 100 μm NADH (D). Plots of rate constants of Cygb‐SH reduction vs. Asc (E) or b5R (F). Rate constants of Cygb‐SH reduction by varied Asc or b5R were obtained from the slopes of the lines in (C) and (D).

Figure 4.

Reduction of three Cygbs (Cygb‐SH, Cygb‐SS, and Cygb‐SC) by varied concentrations of Asc or b5R. Plots of ln(Abs − Abs_b) vs. time as the three Cygbs (~ 3 μm) were reduced by 10 mm Asc (A) or 30 nm b5R in the presence of 0.5 μm b5 and 100 μm NADH (B). Plots of rate constants of 2 μm Cygbs (Cygb‐SH, Cygb‐SS, and Cygb‐SC) reduction by varied Asc concentrations (C) or by varied b5R concentration in the presence of 0.5 μm b5 and 100 μm NADH (D).

O₂‐dependent NO metabolism by modified Cygbs

The Cygb‐mediated O₂‐dependent NO metabolism was examined by simultaneously measuring changes in NO and O₂ concentrations with NO and O₂ electrodes. The rates of NO metabolism in the presence of 0.4 μm Cygbs and 400 μm Asc under varying O₂ concentrations are shown in Fig. 5. The rate of NO metabolism by Cygb‐SS under room air is lower than that by Cygb‐SH and Cygb‐SC. The measured rates of NO decay (V_NO) under room air (at ~ 200 μm [O₂]) are 13.0 ± 1.2 nm·s⁻¹ (Cygb‐SS), 16.1 ± 1.0 nm·s⁻¹ (Cygb‐SH), and 16.7 ± 1.2 nm·s⁻¹ (Cygb‐SC). When O₂ in the solution was gradually decreased, the rate of NO metabolism by the three Cygbs gradually decreased in a similar pattern.

Figure 5.

Plots of V_NO of Cygbs vs [O₂]. Rate of NO metabolism by 0.4 μm of Cygb‐SS, Cygb‐SH, and Cygb‐SC in the presence of 400 μm Asc at different O₂ concentrations.

Discussion

Like other globins, Cygb binds O₂ at the sixth coordination site apical to the heme iron. The intrinsic histidine of Cygb competes with O₂ binding at the sixth coordination site. It has been reported that formation of an intramolecular disulfide bond in Cygb can largely change the equilibrium binding constant of the intrinsic distal histidine 21, which can significantly change the apparent O₂‐binding equilibrium constant. The disulfide bond, if formed, can be reduced by DTT to form two free SH groups. It was reported that the P₅₀ of O₂ binding to Cygb is 1.8 Torr after Cygb is treated with DTT 20, 31, while the P₅₀ of O₂ binding to Cygb without DTT treatment was reported in the range between 0.2 and 1 Torr 3, 20, 32. We tested O₂ binding behaviors for Cygb‐SS (P₅₀ = 0.7 Torr), Cygb‐SH (P₅₀ = 1.4 Torr) and Cygb‐SC (P₅₀ = 2.9 Torr). The measured P₅₀ values for Cygb‐SS and Cygb‐SH (Fig. 2 and Table 1) are in the range of reported values in the literature. Furthermore, we observed that P₅₀ for Cygb‐SC is greater than that for Cygb‐SH.

We then examined the effect of modification of the sulfhydryl group of the cysteines of Cygb on the rate of Cygb reduction. Chemical kinetics with mathematical modeling is a powerful tool to study complicated reaction processes 5, 33, 34, 35. Using this tool, we obtained Eqn (10) for determining the rate constant of Cygb reduction from experimental data of spectrophotometric measurements. It was observed that the rate of Cygb‐SH, Cygb‐SC, and Cygb‐SS reduction by Asc and by b5R/b5/NADH increases with Asc and b5R concentration, respectively. A group of typical experimental curves for the reduction of Cygb‐SH with varying concentrations of Asc (1–20 mm) and b5R (5–120 nm) were shown in Fig. 3A,B. Using Eqn (10), we determined the pseudo first‐order rate constants from the experimental curves in Fig. 3A,B. The data process was demonstrated in Fig. 3C–F. From Fig. 3E,F, we can see that the determined pseudo first‐order rate constants are nearly linear with Asc or b5R concentrations, and the intercepts of the fitted lines are greater than zero. From the slopes of the plots shown in Fig. 4A–D, we can see that the pseudo first‐order rate constants of Cygb‐SH, Cygb‐SC, and Cygb‐SS reduction by Asc and b5R/b5/NADH are close to each other with the rate constant of Cygb‐SS reduction ~ 25% lower than those of Cygb‐SH and Cygb‐SC. Because the rate of Cygb reduction is the rate‐limiting step for NO dioxygenase activity of Cygb 23, the small differences (~ 25%) in rate constants of Cygb reduction after modification of sulfhydryl groups imply that the modification of sulfhydryl groups on the Cygb only cause a small effect on the NO dioxygenase activity of Cygb although this modification more largely shifts the apparent O₂‐binding constant. Correspondingly, the differences in the O₂‐dependent NO dioxygenase activity for the three Cygbs (Fig. 5) parallel the differences in their reduction rates with ~ 25% decrease seen in Cygb‐SS compared to Cygb‐SH and Cygb‐SC (Fig. 4A).

The process of NO dioxygenation requires O₂ binding to Cygb to form Cygb(Fe²⁺O₂) which can rapidly react with NO. If we gradually remove O₂ from the test solution, O₂ concentration in the solution will gradually decrease. Since the modification of sulfhydryl groups of Cygb can shift the P₅₀, differences in the Cygb(Fe²⁺O₂) concentration for the three Cygb preparations may be seen when pO₂ drops below 10 Torr (Fig. 2). From Fig. 2 we see that Cygb‐SS will have the highest Cygb(Fe²⁺O₂) concentration and Cygb‐SC will have the lowest. Thus, under low pO₂ conditions, one could speculate that the NO dioxygenase activity by Cygb‐SS might be the highest among the three Cygbs. However, only a small change in the NO dioxygenase activity was observed for Cygb‐SS compared to Cygb‐SH or Cygb‐SC at O₂ concentrations below 120 μm (Fig. 5). Thus, although the modification of the sulfhydryl groups on the Cygb can largely increase the apparent O₂‐binding constant of Cygbs in the absence of NO, the bound O₂ on ferrous Cygb or Cygb(Fe²⁺O₂) can be immediately removed by NO if NO is present in the solution because NO rapidly reacts with Cygb(Fe²⁺O₂) to form ${\text{NO}}_3^{-}$ and Cygb(Fe³⁺). As a result, almost all of Cygb(Fe²⁺O₂) will react with NO in a short time. The NO dioxygenase activity is then limited by the rate‐limiting step, reduction of Cygb. The modification of the sulfhydryl groups on Cygb does not greatly change the rate of Cygb reduction; therefore, the NO dioxygenase activity is only modestly affected by the modification of the sulfhydryl groups of Cygb.

In summary, the modification of the sulfhydryl groups on Cygb can largely change (~ 4‐fold) the apparent constant of O₂ binding to Cygb. However, this modification only causes a small difference (~ 25%) in the rate constant of Cygb reduction and NO consumption by Cygb. These results indicate that it is possible to largely change one property of Cygb (such as O₂ binding ability) and keep another property of Cygb (such as NO dioxygenase activity) almost unchanged or only modest change.

Author contributions

XL and JLZ supervised the work of this study. AL provided supervision and support for DZ. XL wrote the paper with input from JLZ. DZ performed spectrophotometric and electrochemical experiments. XL and DZ designed experiments and analyzed data with input from JLZ. CH and DZ prepared Cygb samples and wrote the related methods. JB performed PAGE experiments and wrote the related methods. XL, JLZ, CH, JB, DZ, and AL all contributed to the manuscript and its revisions.

Supporting information

Data S1. Equations for determining rate constants of Cygb reduction.