Interaction between Inducible Nitric Oxide Synthase and Calmodulin in Ca2+-Free and -Bound Forms Han Xiao,† Hui Zhou,† Guifang Chen,† Shanli Liu,‡ and Genxi Li*,† Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China, and School of Life Science and Shanghai Key Laboratory of Bio-Energy Crops, Shanghai University, Shanghai 200444, People’s Republic of China Received October 16, 2006
We have obtained the first direct electrochemistry of full-length inducible nitric oxide synthase (iNOS) by entrapping the enzyme in polyethylenimine (PEI) film. The interaction between iNOS and calmodulin (CaM) was then studied, which revealed an enhanced electron-transfer reactivity of the enzyme facilitated by CaM. It was also found that interflavin electron transfer of iNOS could be activated by the binding of Ca2+-bound CaM. The formal potentials (E°′) of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were determined to be -470 and -284 mV vs SCE at pH 7, respectively. The effect of Ca2+ on the interaction between iNOS and CaM has been examined as well. CaM bound with adequate Ca2+ was shown to have a better capability to enhance the electron-transfer reactions within iNOS. Keywords: inducible nitric oxide synthase • calmodulin • electrochemistry • molecule interaction • calcium
1. Introduction Nitric oxide (NO) has firmly established its crucial role in cellular signal transduction considering its important function in a diverse array of physiological processes.1 In vivo, NO is produced by nitric oxide synthase (NOS), which catalyzes the oxidation of L-arginine to citrulline and NO with NADPH and O2 as cosubstrates.2 The currently known NOS family has three isoforms on the basis of their constitutive (eNOS and nNOS) versus inducible (iNOS) expression, all containing tightly bound cofactors of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and iron protoporphyrin (heme).3 The active form of the NOS isoforms exists as a homodimer with a calmodulin (CaM)-binding sequence linking the flavins domain to the heme domain with a binding site for 5,6,7,8-tetrahydrobiopterin (H4B).4,5 Because of the importance of NO in cellular signaling, much attention has been given to the study of the NOS isoforms. Although many issues regarding the details of NO synthesis have far from been elucidated, it is generally thought that the three NOS isoforms catalyze the same two-step oxidation and appear to have the same catalytic mechanism.6 L-Arginine is oxidized to the stable intermediate N-hydroxy-L-arginine and subsequently to NO and citrulline. During these reactions, electrons from NADPH are shuttled through FAD to FMN, and finally to the heme.7,8 Therefore, it might be possible to conduct studies by using an electrochemical technique to study these biological macromolecules.9-16 Here, in our work, we first utilize * Corresponding author. Tel.: (+86)(25) 3593596. Fax: (+86)(25) 3592510. E-mail:
[email protected]. † Nanjing University. ‡ Shanghai University.
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an electrochemical method to investigate the direct electrochemistry of full-length iNOS, which is the first report of this kind to our knowledge. By embedding iNOS in a polyethylenimine (PEI) film, its electrochemical response can be observed at the PEI-modified pyrolytic graphite (PG) electrode. CaM, a ubiquitous Ca2+-binding protein taking part in a wide variety of signaling pathways, is the first protein demonstrated to interact with NOSs.17 All three NOS isoforms are CaM dependent for the enzymatic activity of NO synthesis.18 CaM binding has been proven to be able to increase the rate of electron transfer from NADPH to the two flavins of reductase domain and also trigger the electron transfer from the reductase domain to the heme center.19-21 Ca2+-dependence may distinguish iNOS from eNOS and nNOS, because CaM is bound to iNOS in a seemingly irreversible manner,22 while the latter two isoforms, which bind CaM reversibly, have higher Ca2+ concentration requirements.23 In this paper, we report our findings that the binding of CaM with iNOS is able to enhance the electron-transfer reactivity of the enzyme. Furthermore, electron transfer between the interflavin redox couples of iNOS is found to be achieved when the enzyme is bound by Ca2+bound CaM. This suggests that CaM bound with Ca2+ will facilitate the electron transfer between the two flavins to a greater extent.
2. Experimental Section Materials. Buffered aqueous solution of recombinant mouse iNOS (EC 1.14.13.39, expressed in Escherichia coli, no. N2783) was obtained from Sigma. CaM (EC number 2773707, from bovine brain, no. P0809), FAD disodium salt hydrate (no. 6625), FMN disodium salt dihydrate (no. F8399), and PEI were also purchased from Sigma and used without further purification. 10.1021/pr060544l CCC: $37.00
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Inducible Nitric Oxide Synthase and Calmodulin
Other chemicals were of analytical reagent grade. Water was purified with a Milli-Q purification system (Barnstead, MA) to a specific resistance >18 MΩ cm and was used to prepare all solutions. iNOS was stored in single-use aliquots at -70 °C. CaM was dissolved in a 0.2 M, pH 7 phosphate buffer. Solutions of FAD, FMN, and PEI were prepared as 0.5 mg mL-1, 5 mM, and 1%, respectively. Electrode Preparation. The PG electrode was prepared by putting a PG rod into a glass tube and fixing it with epoxy resin. Electrical contact was made by adhering a copper wire to the rod with the help of wood alloy. Prior to surface modification, the PG electrode surface was first polished on rough and fine abrasive papers. Next, the surface was polished to mirror smoothness with an alumina (particle size of about 0.05 µm)/ water slurry on silk. Finally, the electrode was thoroughly washed through ultrasonicating in both ethanol and double distilled water for about 5 min. A mixture containing 10 µL of 7.5 mg mL-1 iNOS and 10 µL of PEI was deposited on the PG electrode surface followed by slow drying overnight and was thoroughly rinsed with pure water before use. Electrochemical Measurements. Cyclic voltammetric measurements were performed using a PARC 263A potentiostat/ galvanostat (EG&G; Princeton, NJ) and a three-electrode system. A one-compartment glass cell with a modified PG working electrode, a saturated calomel reference electrode (SCE), and a platinum wire auxiliary electrode was used for the measurements, with a working volume of 5 mL. The buffer solution was a 0.2 M phosphate buffer solution (PBS), pH 7.0. It was bubbled thoroughly with high-purity nitrogen for about 5 min prior to experiments. The anaerobic environment was maintained throughout by keeping nitrogen over the surface of the solution thereafter. All experiments were carried out at ambient temperature of about 20 °C.
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Figure 1. CVs obtained at iNOS/PEI-modified PG electrode (-); PEI alone-modified PG electrode (- - -); and bare PG electrode in an O2-free phosphate buffer solution, 0.2 M, pH 7.0 (‚ ‚ ‚). Scan rate: 200 mV/s. Inset: Plot of the peak current versus scan rate.
3. Results and Discussion Intramolecular electron flow in iNOS dimer has been known to start with the two-electron oxidation of NADPH. The electron goes via FAD to FMN in the reductase of one monomer, then to the heme iron in the oxygenase domain of the second monomer. In our protocol, the PG electrode replaces NADPH as the electron donor, with the imposed potential, triggers the electron-transfer reaction. Figure 1 shows a typical cyclic voltammogram (CV) of iNOS confined in the PEI film on a PG electrode surface. A pair of well-defined and reproducible peaks can be observed in an O2-free PBS at pH 7.0. The formal potential (E°′) is about -470 mV. The peak separation is 52 mV at the scan rate of 200 mV/s, indicative of a fast, reversible electrochemical process (59 mV).24 In contrast, no response can be obtained from either the bare PG electrode or the substrate electrode modified alone with PEI under the same conditions. Thus, it is reasonable to assign the peak couple to the FAD redox center, and PEI is a good material to provide an ideal microenvironment for the achievement of direct electron transfer from the electrode to iNOS. The effect of scan rate on the response of the immobilized iNOS is shown in the inset of Figure 1. Both the cathodic and the anodic peak currents (Ipc and Ipa) are linear with scan rate ranging from 50 to 700 mV, which is characteristic of thin-layer electrochemical behavior.24 Electron transfer in NOSs is known to be proton-coupled. So, we have examined this issue. Figure 2 shows the influence of pH on the redox peak couple of iNOS. Stable and welldefined peaks can be obtained in the pH range of 4-10, and
Figure 2. CVs obtained at iNOS/PEI-modified PG electrode in 0.2 M phosphate buffer solution with pH 5.8, 7.0, and 8.4 (from left to right). Others same as in Figure 1. Inset: Plot of the formal potential versus pH value.
E°′ shifts negatively with increased pH. E°′ shows a linear dependency upon pH (inset in Figure 2), with a slope of -45 mV/pH unit, close to the data for a one-proton reduction process.25 CaM is believed to bind to an amino acid sequence located approximately midway between the two flavins of reductase domain and to play a crucial role in controlling the electrontransfer activity of all NOS isoforms. The effect of CaM on iNOS was studied. First, we add CaM in the test solution for the electrochemical experiments of iNOS. No change can be observed concerning the response of iNOS. The reason might be the lack of possible access for CaM to the binding site in iNOS, because the enzyme is confined in PEI film. So, we adopted the strategy of “co-modification” of CaM and iNOS at the electrode surface to maximize the interaction between CaM and iNOS. Prior to the electrode modification, iNOS was first incubated with CaM at different concentrations. Next, the substrate electrode was modified with the mixture of PEI and CaM-bound iNOS, whose final concentration was controlled as previously discussed. Journal of Proteome Research • Vol. 6, No. 4, 2007 1427
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Figure 3. Part of CVs obtained at CaM-bound iNOS/PEI-modified PG electrode in an O2-free phosphate buffer solution, 0.2 M, pH 7.0. Scan rate: 200 mV/s. Background subtracted.
Xiao et al.
Figure 5. CVs obtained at CaM-bound iNOS/PEI-modified PG electrode with different scan rate from 50 to 700 mV/s. CaM (1 mg/mL) has been incubated with 0.01 M Ca2+. Inset: Plot of the peak current versus scan rate for FMN center (A) and FAD center (B). Others same as in Figure 4. Scheme 1. Redox Potentials and Direction of Electron Flow from the Electrode to Flavins Domain of iNOS
Figure 4. CVs obtained at CaM-bound iNOS/PEI-modified PG electrode. CaM (1 mg/mL) has been incubated with 0.01 M Ca2+. Others same as in Figure 3.
Figure 3 shows that the redox peak current of iNOS that has been preincubated with CaM is much higher than that of the enzyme without the treatment of CaM. Also, more CaM makes the peak higher. Further studies reveal that CaM is not electroactive either at the bare PG electrode or by being entrapped in PEI film. Therefore, it is reasonable to attribute this enhanced electron-transfer reactivity of iNOS to the interaction between CaM and iNOS. E°′ for CaM-bound iNOS is also observed to be exactly the same as that of CaM-free iNOS. The reason might be that CaM binding has little effect on the redox potential of NOS but acts purely as a conformation effector.10 We have also studied the effect of Ca2+ on the interaction between CaM and iNOS, which has been given more and more research interest.26-29 Employing the above strategy, we replace CaM with the Ca2+-bound form to examine the effect of the Ca2+ toward the interaction between CaM and iNOS. Prior to the preparation of CaM-bound iNOS, 1 mg/mL CaM is first incubated with Ca2+ (in the form of CaCl2 solution) at a volume ratio of 1:1. Electrochemical studies reveal that iNOS combined with Ca2+-bound CaM will exhibit different electrochemical behavior, as shown in Figure 4. Besides the former redox couple at -470 mV (E1) assigned to the FAD center, a new pair of peaks appear at -284 mV (E2), which should be attributed to the 1428
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redox reaction of another redox center, FMN. The peak separation for E2 is 40 mV at a scan rate of 200 mV/s, which is a little lower than that of E1. The peak currents of the two redox couples are both linear with the scan rate in the test range of 50-700 mV, as shown in Figure 5 and the inset. Electrochemical investigations of free FMN and FAD in PEI film have been conducted for comparison. E°′ values for FMN and FAD are -455 and -458 mV, respectively, both in agreement with the previous report for the two flavins.30 We should notice that the potential of the FMN center in our study is -284 mV, much different from the free FMN as -455 mV. In our system, electron transfer is assumed to take place within iNOS. The explanation could be the electron transfer is easier to achieve from FAD to FMN than that from electrode to FMN. This result is consistent with our protocol and also excludes the possibility that the cofactor may dissociate from the enzyme. So, we can attribute the two redox couples for iNOS to the interflavin electron transfer. The redox potentials and the direction of the electron flow of the whole reductase domain of iNOS are illustrated in Scheme 1. Further studies reveal that Ca2+ alone has no effect on iNOS if it is not bound with CaM. It is of peculiar interest that CaM preincubated with different concentrations of Ca2+ will make a difference to the activity of the FMN redox center of iNOS. If the concentration of Ca2+ is below 10-3 M, Ca2+-bound CaM cannot transfer electrons from FAD to FMN. As the result, “E2” can be hardly observed, while “E1” still exists. When the concentration of Ca2+ is adequate to bring forth both “E1” and “E2” simultaneously, adding more Ca2+ does not make any change either to increase the peak current or to induce new redox peaks. Previous studies suggest that CaM has four Ca2+ binding sites and adopts a different spatial structure when chelated with Ca2+.31 The role of each of the four Ca2+-binding sites of CaM in the activity of iNOS has been proven to be different.26,28 This
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Inducible Nitric Oxide Synthase and Calmodulin
may to some extent explain the experimental results in this work. If the concentration of Ca2+ is low, the crucial binding sites may be Ca2+-free so that CaM may not adopt the required conformation to fully activate iNOS. On the other hand, if the binding site to activate iNOS has been bound to Ca2+, more Ca2+ is not necessary. Furthermore, at higher concentrations of Ca2+ where all of the binding sites of CaM have been occupied with Ca2+, more Ca2+ means nothing. Certainly, the mechanism of Ca2+ in regulating the interaction between iNOS and CaM needs more studies to be clearly elucidated. Nevertheless, our work has firmly demonstrated that CaM bound with adequate Ca2+ can better facilitate the electron transfer within iNOS. In summary, we have studied the interaction between iNOS and CaM in Ca2+-free and -bound forms with electrochemical techniques. By employing an iNOS/PEI-modified electrode, we have obtained information about the electron-transfer activity of iNOS modulated by CaM. This work has also demonstrated the importance of Ca2+ in the interaction between CaM and iNOS, supporting the fact that CaM bound with adequate Ca2+ can have a better capability to enhance the electron-transfer reactions within iNOS.
Acknowledgment. This work is supported by the National Natural Science Foundation of China (Grant Nos. 90406005, 20575028) and the Program for New Century Excellent Talents in University, the Chinese Ministry of Education (NCET-04-0452). References (1) Culotta, E.; Koshland, D. E. Science 1992, 258, 1862-1865. (2) Marletta, M. A. Nitric oxide synthase: aspects concerning structure and catalysis; Cell: Cambridge, MA, 1994; Vol. 78, pp 927-930. (3) Alderton, W. K.; Cooper, C. E.; Knowles, R. G. Biochem. J. 2001, 357, 593-615. (4) Marletta, M. A. J. Biol. Chem. 1993, 268, 12231-12234. (5) Masters, B. S. S. Annu. Rev. Nutr. 1994, 14, 131-145. (6) Ortiz de Montellano, P. R.; Nishida, C.; Rodriguez-Crespo, I.; Gerber, N. Drug Metab. Dispos. 1998, 26, 1185-1189. (7) Marletta, M. A. Chem. Res. Toxicol. 1988, 1, 249-257. (8) Stuehr, D. J. Annu Rev. Pharmacol. Toxicol. 1997, 37, 339-359. (9) Garnaud, P. E.; Koetsier, M.; Ost, T. W. B.; Daff, S. Biochemistry 2004, 43, 11035-11044.
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