Supporting Information

Differential Calmodulin-Modulation and Electron Transfer Properties of Muscle-Specific Slice Variant nNOSµ Compared to nNOSα

Satya P. Panda1, Wenbing Li2, Priya Venkatakrishnan1, Li Chen2, Andrei V. Astashkin3, Bettie Sue S. Masters1, Changjian Feng2, Linda J. Roman1

1Department of Biochemistry, University of Texas Health Science Center in San Antonio, San Antonio, TX 78229

2Department of Pharmaceutical Sciences, University of New Mexico, Albuquerque, NM 87131

3Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721

Figure S1a below shows the absorption spectra of as-isolated nNOSα and nNOSµ proteins. Heme is primarily in the high spin state after treatment with H4B and L-arginine during preparation as indicated by the Soret maximum near 400 nm. Figure S1b shows the EPR spectrum of nNOSµ replete with L-arginine and H4B. The high spin features at g = 7.63, 4.11, and 1.82 are very similar to those of the majority species of the L-Arg- and H4B-replete nNOSα. There is a minor g = 6 axial ferriheme signal from the heme sites that have lost their thiolate ligand. The relative amount of these unliganded sites can be approximately estimated using numerical simulations of the EPR spectra as ~1% only. There is also a detectable FMNH radical in the EPR spectrum (the narrow line at g = 2). In addition, we measured the fluorescence spectra of the nNOSµ and nNOSα proteins under identical conditions, i.e., same protein concentration, buffer, and fluorometer settings (Figure S2). CaM binding to nNOSµ increases fluorescence, similar to nNOSα, while the CaM-bound nNOSµ protein exhibits slightly higher flavin fluorescence compared to nNOSα.

(a)

(b)

Figure S1. Characterization of purified nNOS and nNOSµ by UV-vis and EPR spectroscopy. (a) The absorption spectra of purified protein from 300 nm to 900 nm showing a heme Soret peak at 397 nm and flavins in the 450 nm region. Protein concentrations are 7 M each. (b) EPR spectra of nNOS protein, taken under microwave frequency, 9.343 GHz; microwave power, 2 mW; modulation amplitude, 5 G; modulation frequency, 100 kHz; temperature, 6 K. nNOSµ (40 M) in 10mM Tris-HCl, pH 7.4, 100mM NaCl, 1mM L-Arg, 0.1mM DTT, 0.1mM EDTA and 10% glycerol was frozen quickly in cold isopentane, and kept at 77 K until utilized. Continuous wave EPR spectra were recorded on a Bruker Elexsys E500 spectrometer at 6K.

Figure S2. Flavin fluorescence spectra of the nNOS and nNOS proteins. The NOS flavin fluorescence spectra were measured on a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies) at room temperature. The nNOS protein in degassed buffer (40mM Bis-Tris propane, 400mM NaCl, 2mM L-Arg, 10% glycerol, pH 7.6) was passed through a 0.2 m filter. The protein concentrations were 1 M each, and the measurements were performed under the same conditions; excitation wavelength: 446 nm. Data are representative of two experiments.

(nNOSµ) (nNOSα)

Figure S3. Rates of NO production at different CaM concentration with varying L-Arg concentration. The NOS activity was measured at different CaM concentrations with varying L-Arg concentrations (0.25 – 10 µM). The apparent Km for L-Arg was measured for each CaM concentration by fitting the data to the Michaelis-Menten equation.

Figure S4: Rates of flavin reduction using rapid kinetics measurements. The rates of flavin reduction were measured for both nNOS and nNOSµ in the absence and presence of Ca2+/CaM as the change of absorbance at 485 nm using stopped-flow spectrophotometry. Traces show the observed kinetics (open circles) and the calculated fit (solid line). Residuals for each fit are shown below the kinetic traces.

(a)

(b)

Figure S5. Transient trace at 460 nm at (a) 0 0.2 s and (b) 0 4 s obtained for the [Fe(II)-CO][FMNH] form of nNOS flashed by a 446 nm laser. Sample temperature was set at 21 C. Anaerobic solution contained ~ 10 M NOS protein, ~ 20 M dRF and 5 mM fresh semicarbazide in pH 7.6 buffer (40 mM Bis-Tris propane, 400 mM NaCl, 2 mM l-Arg, 20 M H4B, 1 mM Ca2+ and 10 % glycerol). Solid line corresponds to the best single-exponential fit to the data.

(nNOSµnNOS)

Figure S6: Rate of ferrous nitrosyl complex formation under turnover condition using rapid kinetics measurements. Rates of ferrous nitrosyl complex formation were measured for both nNOS and nNOSµ under turnover conditions, i.e. in the presence of L-Arg, H4B, Ca2+/CaM and NADPH, as a change in absorbance at 436nm using stopped-flow spectrophotometry. Traces show observed kinetics (open circle) and the calculated fit (solid line).

Table S1. The FMN−heme IET rate constants ket of nNOS and nNOS proteins over temperature range from 283 to 304 K.a

Temperature (K)

ket (s-1)

nNOS

nNOS

283.15

27.9 0.5

33.8 0.3

288.15

29.7 0.6

41.5 0.4

294.15

33.4 0.4

54.5 0.5

299.15

50.8 0.8

82.8 0.8

304.15

64.2 1.6

103 1.3

a Buffer: 40 mM Bis-Tris propane, 400 mM NaCl, 2 mM l-arginine, 20 M H4B, 1 mM Ca2+, 10 % glycerol, pH 7.6. Final nNOS protein concentration: ~ 10 M. The FMN−heme IET kinetics was monitored at 460 nm. The rates values are average from at least two independent experiments.

S1