Is Nostoc H-NOX a NO sensor or redox switch?

Abstract

Nostoc sp. (Ns) H-NOX is a heme protein found in symbiotic cyanobacteria, which has approximately 35% sequence identity and high structural homology to the beta subunit of soluble guanylyl cyclase (sGC), suggesting a NO sensing function. However, UV-vis, EPR, NIR MCD, and ligand binding experiments with ferrous and ferric Ns H-NOX indicate significant functional differences between Ns H-NOX and sGC. (1) After NO binding to sGC, the proximal histidine dissociates from the heme iron, causing a conformational change that triggers activation of sGC. In contrast, formation of pentacoordinate (5c) NO heme occurs to only a limited extent in Ns H-NOX, even at >1 mM NO. (2) Unlike sGC, two different hexacoordinate (6c) NO complexes are formed in Ns H-NOX with initial and final absorbance peaks at 418 and 414 nm, and the conversion rate is linearly dependent on [NO], indicating that a second NO binds transiently to catalyze formation of the 414 nm species. (3) sGC is insensitive to oxygen, and ferric sGC prepared by ferricyanide oxidation has a 5c high-spin heme complex. In contrast, Ns H-NOX autoxidizes in 24 h if exposed to air and forms a 6c ferric heme complex, indicating a major conformational change after oxidation and coordination by a second histidine side chain. Such a large conformational transition suggests that Ns H-NOX could function as either a redox or a NO sensor in the cyanobacterium.

Figure 1

Kinetics of autooxidation of ferrous Ns H-NOX. 6 µM of ferrous Ns H-NOX in the absence of DTT was placed in the HP8453 diode array spectrophotometer to follow the optical changes up to 2 days at 24 °C. The kinetics of ΔA_409−443 was fit to one-exponential function to obtain the rate of autooxidation (Inset).

Figure 2

A: Kinetics of CO binding to the ferrous Ns H-NOX. Ferrous Ns H-NOX, 5 µM, prepared in an anaerobic tonometer was reacted with CO in a stopped-flow at 24 °C. CO was prepared in anaerobic buffer at concentrations varying from 25 to 166 µM. One-exponential change of absorbance at 420 nm was used to determine the observed rates and used for calculation of the 2^nd-order on rate constant (Inset). Two sets of experiments using two different batches of sensor protein were conducted (red and black circles). Standard deviation of at least triplicates at each concentration was shown by the significant bar. B: Determination of the dissociation rate constant of CO binding. CO-ferrous Ns H-NOX complex was pre-formed by mixing 4 µM sensor with 1 mM CO in an anaerobic glass tonometer, and then reacted with 1 mM NO solution. The CO-dissociation rate was followed by diode array or single wavelength stopped-flow at 422 nm (Inset) for 8s data collection at 24 °C. Kinetic data were fit to 2- exponential function.

Figure 2

A: Kinetics of CO binding to the ferrous Ns H-NOX. Ferrous Ns H-NOX, 5 µM, prepared in an anaerobic tonometer was reacted with CO in a stopped-flow at 24 °C. CO was prepared in anaerobic buffer at concentrations varying from 25 to 166 µM. One-exponential change of absorbance at 420 nm was used to determine the observed rates and used for calculation of the 2^nd-order on rate constant (Inset). Two sets of experiments using two different batches of sensor protein were conducted (red and black circles). Standard deviation of at least triplicates at each concentration was shown by the significant bar. B: Determination of the dissociation rate constant of CO binding. CO-ferrous Ns H-NOX complex was pre-formed by mixing 4 µM sensor with 1 mM CO in an anaerobic glass tonometer, and then reacted with 1 mM NO solution. The CO-dissociation rate was followed by diode array or single wavelength stopped-flow at 422 nm (Inset) for 8s data collection at 24 °C. Kinetic data were fit to 2- exponential function.

Figure 3

A: Optical characterization of Ns H-NOX binding to stoichiometric and excess amount of NO. UV-Vis spectral changes recorded by anaerobic rapid-scan diode array stopped-flow within 1 s or 2 min for 5 µM Ns H-NOX reaction with 1:1 and 5:1 ratio of NO, respectively, at 24 °C. B: Kinetics of Ns H-NOX binding with excess NO under pseudo first-order conditions. 5 µM ferrous Ns H-NOX was mixed with > 10 × of NO at different concentrations and the kinetics was recorded by single wavelength stopped-flow at 428 nm at 50 ms/ 5s split time mode to cover the triphasic changes. Data were fit by 3-exponential function and the first fast phase that show [NO] dependence was further analyzed to obtain the 2^nd order binding rate constant as the secondary plot (Inset). Average of three shots or more with the standard deviation plotted as the error bar for each NO concentration in two different sets of experiments (blue and red symbols). The linear regression (red dash) and the slope and y-intercept obtained are that with one of the two sets of data.

Figure 3

A: Optical characterization of Ns H-NOX binding to stoichiometric and excess amount of NO. UV-Vis spectral changes recorded by anaerobic rapid-scan diode array stopped-flow within 1 s or 2 min for 5 µM Ns H-NOX reaction with 1:1 and 5:1 ratio of NO, respectively, at 24 °C. B: Kinetics of Ns H-NOX binding with excess NO under pseudo first-order conditions. 5 µM ferrous Ns H-NOX was mixed with > 10 × of NO at different concentrations and the kinetics was recorded by single wavelength stopped-flow at 428 nm at 50 ms/ 5s split time mode to cover the triphasic changes. Data were fit by 3-exponential function and the first fast phase that show [NO] dependence was further analyzed to obtain the 2^nd order binding rate constant as the secondary plot (Inset). Average of three shots or more with the standard deviation plotted as the error bar for each NO concentration in two different sets of experiments (blue and red symbols). The linear regression (red dash) and the slope and y-intercept obtained are that with one of the two sets of data.

Figure 4

Determination of the association (A) and dissociation (B) rate constants of NO binding to Ns H-NOX for formation of the 1^st 6c NO complex by flow-flash and stopped-flow. A: CO complex of Ns H-NOX was first prepared with 50 µM sensor protein and 100 µM CO anaerobically in a syringe. This complex was briefly mixed with 1 mM NO in the stopped-flow for 50 ms and a 0.5 µs 577 nm dye laser was applied to flash off the bound CO. Subsequent NO association kinetics was monitored at both 436 (red trace) and 418 nm (blue trace). Black lines are one-exponential fittings for both data. B: NO complex of Ns H-NOX was first prepared anaerobically by mixing 5 µM sensor protein with 10 µM NO in a glass tonometer. This mixture was then mixed with a mixture of 1 mM CO and 25 mM dithionite and aged for 50 ms in a rapid scan stopped-flow and the electronic absorption spectra at 3 ms (black) and 270 s (blue) were shown together with that at zero time. Single wavelength kinetic data at 428 and 400 nm with opposite amplitude changes are shown in the Inset and fit to 1-exponential function to obtain the rate constant. All reactions were conducted at 20 °C.

Figure 4

Determination of the association (A) and dissociation (B) rate constants of NO binding to Ns H-NOX for formation of the 1^st 6c NO complex by flow-flash and stopped-flow. A: CO complex of Ns H-NOX was first prepared with 50 µM sensor protein and 100 µM CO anaerobically in a syringe. This complex was briefly mixed with 1 mM NO in the stopped-flow for 50 ms and a 0.5 µs 577 nm dye laser was applied to flash off the bound CO. Subsequent NO association kinetics was monitored at both 436 (red trace) and 418 nm (blue trace). Black lines are one-exponential fittings for both data. B: NO complex of Ns H-NOX was first prepared anaerobically by mixing 5 µM sensor protein with 10 µM NO in a glass tonometer. This mixture was then mixed with a mixture of 1 mM CO and 25 mM dithionite and aged for 50 ms in a rapid scan stopped-flow and the electronic absorption spectra at 3 ms (black) and 270 s (blue) were shown together with that at zero time. Single wavelength kinetic data at 428 and 400 nm with opposite amplitude changes are shown in the Inset and fit to 1-exponential function to obtain the rate constant. All reactions were conducted at 20 °C.

Figure 5

EPR spectra of Ns H-NOX at different redox state and coordination. EPR spectra are presented (from top to bottom) for 80 µM isolated ferrous Ns H-NOX, 55 µM ferric Ns H-NOX prepared by ferricyanide oxidation and gel-filtration chromatography, 50 µM ferric Ns H-NOX mixed with 50 mM imidazole, and ferrous NO complex formed with 160 µM NO. EPR conditions were: 4 mW, 9.602 gHz, time constant: 0.33s, modulation amplitude: 10.9G and 10K.

Figure 6

EPR characterization of the ferrous NO complex of Ns H-NOX. 70 µM ferrous Ns H-NOX was reacted with 2:1 NO by injecting 2 mM NO-saturated buffer under anaerobic condition and manually frozen for EPR assessment (bottom solid trace). The hyperfine splitting by NO nitrogen nucleus and super hyperfine splitting by the proximal histidine were indicated for the g_z component as well as the isotropic hyperfine splitting for the g_y and g_x component optimized via spectral simulation to the dominant 6c low-spin NO complex (bottom dashed trace). An EPR for 60 µM ferrous NO complex of M144I sensor dominated by 5c NO complex was juxtaposed for comparison (top solid trace). The three g values and the associated hyperfine parameter values for a simulated 5c-NO complex spectrum (top dashed trace) that closely matches the acquired spectrum are also indicated for comparison with those obtained for the wild type sensor. Progressive power saturation at LN temperature, the value of half-saturation power, P_1/2, and value of b by fitting the data to equation (2) are also shown for the wild type sample (Inset). EPR conditions were the same as described in Fig. 5 except 1.87 G modulation amplitude and 9.28 gHz frequency.

Figure 7

Truth diagram analysis of the low-spin ferric Ns H-NOX and its imidazole complex. Correlation between the heme rhombicity and the axial ligand strength of many low-spin heme complexes (various symbols) are mapped in six different zones. Complexes in five of them have histidine as proximal ligand and with cyanide (zone CN−), methionine (zone C), histidine (zone B), histidinate/azide (zone H), and hydroxide/phenolate (zone O) as the distal ligand. Those hemeprotein containing a cysteine thiolate proximal ligand including P450, chloroperoxidase and nitric oxide synthase and their derivatives fall in the P zone. This diagram was modified from the original Blumberg Peisach convention (33, 51). The grey solid circles are the ferric Ns H-NOX and its imidazole derivative.

Figure 8

Candidate axial heme ligand for ferric Ns H-NOX. Potential heme-ligand residues nearby heme iron and at the distal heme side of the wild type ferrous Ns H-NOX are displayed. Distances between two candidate methionine residues (Met144 and Met1), N-terminal amino group, Trp74 indole nitrogen and the heme iron (orange ball) are given. The second closest histidine, His150 and the closest lysine, Lys7 and the proximal heme ligand, His105, are also shown in stick model. Heme porphyrin is shown as red stick model. This is a RASMOL representation of PDB identifier: 2O09.

Figure 9

Similarity of EPR spectrum between Ns H-NOX and cytochrome c. EPR of 65 µM ferric Ns H-NOX (red), 30 µM M144I Ns H-NOX (blue), and 500 µM bovine cytochrome c (black) are directly compared for the position of the principal g values of the low-spin rhombic heme. The additional features including the high-spin heme at g = 6 region and the non-specific iron at g = 4.3 and organic radical at g = 2 present in the Ns H-NOX are not present in cytochrome c. The two broad features highlighted by the red stars are artifacts of the resonator. The EPR amplitude for the M144I mutant has been adjusted for its concentration difference from the wild type sample.

Figure 10

Identification of the axial heme ligands of ferric Ns H-NOX by NIR MCD. Same three samples used for EPR measurements in Fig. 9 were used in the NIR MCD measurements in the range from 800 to 2000 nm at 24 °C. All samples were prepared in D₂O 50 mM HEPES, pH 7.4 to minimize the NIR MCD overtone background signals from the water solvent (34). MCD conditions are described in the Methods and the spectrum for cytochrome c was obtained as one scan while that of wild type and M144I Ns H-NOX were an average of 8 scans. The wavelengths of the main transitions are labeled for each sample. These marker wavelengths (nm), or energy levels are used to identify the axial ligand pairs from the correlation diagram composed for a series of low-spin hemeproteins with two axial ligands (34).

Figure 11

Far-UV CD of the ferric and ferrous Ns H-NOX. Both the ferrous and ferric sensor proteins were prepared as 1 µM in 2 mM HEPES containing 2 mM NaCl and 0.1 % glycerol, pH 7.7 by either dithionite or ferricyanide pre-treatment, respectively, and then gel-filtered in the same HEPES buffer to remove excess reductant or oxidant. CD measurements were conducted as described in the Experimental Procedures. The data shown for the ferric (black), ferrous (red) sensor are 12-scan average corrected for background signal originated from buffer alone and the difference spectrum (blue) is obtained by subtracting the CD spectrum of the ferrous sensor from that of the ferric form.

Figure 12

Equilibrium and kinetic binding of cyanide and azide to the ferric Ns H-NOX. To build binding isotherm for cyanide and azide binding (A), 2.9 µM ferric Ns H-NOX was titrated by aliquots of 0.2 M cyanide to the final concentration of 5 mM (black circles); and 5.8 µM ferric sensor protein was titrated with 1M azide to 3 mM final concentration (red circles), respectively, to reach saturation. Maximal absorbance changes at 307 – 406 nm for cyanide heme complex formation and 377 – 417 nm for azide heme complex formation at different ligand concentrations were fit by single binding site saturation hyperbola (equation 1) (solid lines). The volume change by addition of either ligand was less than 2.5% and was not corrected further for calculating the free ligand concentrations. For kinetic binding measurements (B), 2.9 µM ferric Ns H-NOX was mixed with a series of ligand concentrations at least 10 fold excess to the protein and the absorbance changes at 430 nm in a stopped-flow. The observed rates obtained by single (for azide, red circles) and double (for cyanide, black and blue circles) exponential function were plotted against ligand concentrations. Solid line is the fit to hyperbolic function to show [CN⁻]- dependent saturation represented by equation 1. Error bars are the standard deviation of triplicate shots. Reaction temperature for both equilibrium and kinetic binding experiments was 24 °C.

Scheme I

Scheme II