Horse Heart Cytochrame c and to Its Carboxymethylated Derivative: A Spec&wcupk and Thermodynamic Study

Paolo Ascenzi, Massimo Coletta, Roberto Santucci, Francesca Polizio, and Alessandro Desideri

PA. Department of Pharmaceutical Chemistry and Technology, University of Turin, and Department of Biochemical Sciences “Alessandro Rossi Fanelli,” Universig qf Rome “La Sapienza’.- MC, RS. Department of Molecular, Cellular and Animal Biology, University of Camerino.--FP. Department of Biology, University of Rome “Tor Vergata”.-AD. Department of Organic and Biological Chemistq Univers#y of Mekna, Italy.

Nitric oxide binding to ferrous native horse heart cytochrome c and to its carboxymethylated derivative fias been Invest&red quantirarivehj by EPR and absorbance spec&oswpF The X-band EPR spectra and the absorption spectra in the Soret region of the nitrosylated derivative of ferrous native and carboxymethylated cytochrome c display the same basic characteristics reported for the beef heart cytochrome a3 in cytochrome c oxidase, and horseradish and baker’s yeast cytochrome c peroxidase, as well as the high affinity form of oxygen carrying proteins. Values of the dissociation equilibrium constant for nitrosylation of ferrous native and carboxymethylated cytochrome c are 8.2 x lop6 M and I 5 x lo-* M, respectively, at pH 7.0 and 10°C. The results here reported represent clearcut evidence for the nitric oxide-induced cleavage of the Fe-Met80 bond in ferrous native cytochrome c, and allow estimation of the free energy associated to the heme-iron sixth coordination bond (> 10 kJ mol-‘, at 1OT).

Address reprint requests and correspondence to: Professor Paolo Ascenzi, Department of Biochemical Sciences “Alessandro Rossi Fanelli,” University of Rome “La Sapienza,” Piazzale Aldo Moro 5, 00185 Rome, Italy.

Journal of Inorganic Biochemistry, 53,273-280 (1994) 273 0 1994 Elsevier Science Inc., 655 Avenue of the Americas, NY, NY 10010 0162-0134/94/%7.00

NITRIC OXIDE BINDING TO CYTOCHROME c 275

oxide &SiVa%Ye Of ?fenDm B~YLZ mh cxb~mBh$.aJ8~ pm&~Dm~ J _!&2npb&

obtained by the different methods, were superimposable. The concentration of native cytochrome c was determined on the basis

of E= 11.1 mM_’ cm-’ (at 530 nm), 131.2 mM_’ cm-’ (at 415 nm) and 120.8 mM --” cm -’ Jat 432 nn$ for tie f?zr_+ f%z~oq sod r~_-ii_xrz# d~~ti@_jYq respcective@ )see Ref. 13 anb Fig, 1, panti A’> me CDBcmG--abDn ~3 Jh?

carbqyme%y>a’rer5 beim~~~ DE cyZDch~~me .c was h%zoiv_w~ DD Bx ha&s

of E = 9.4 mM_’ cm- ’ (at 530 nm), 106.0 mM_’ cm- ’ (at 415 nm) and 120.3mm + cm -” i’at 4i+,T nm j rbr the tirric, tirrous, and’ nitrosyl’ derivatives, resptetirve)y )see Ref. 2 anb Fjg 2, panes a’>, YAues Djnahz aB3 C&D+&)-

ylated cytochrome c concentration obtained from values of E for the different ferric a& ferrous Aer%&jyes are m exc&e,r agxxmcnl I > 99%)

GaV%rAY &?ii~ “*?I% <“&Q> JJaYY u*zri-n& ?roIrr Aii.?I-i& zh-em-&? &. (Milwaukee, WI, U.S.>, and potassium nitrite (K15NOJ was purchased from Cambridge Isotope Laboratories (Woburn, MA, U.S.). Sephadex G-25 was obtained from Pharmacia LKB Biotechnology Sverige AB (Bromma, Sverige). All ether praducs were purc(lasecl fram 8&r& AG (Oarms&&, German..). tic chemicals were of analytical grade and used without further purification.

Nitric oxide binding to native cytochrome c and to its carboxymethylated derivative was followed by EPR and absorbance spectroscopy.

$pkJ ;jfJ m :lolf-J 320 335 350 410 440

Magnetic Field (mT) il (nm) FIGURE 1. X-band EPR spectra (panels A and B) and absorption spectra in the Soret region (panels A’ and B’) of the nitric oxide derivative of ferrous native (panels A and A’) and carboxymethylated (panels B and B’) cytochrome c. X-band EPR spectra were obtained upon reaction of l4 NO and “NO to ferrous native and carboxymethylated cytochrome c (spectra a and b, respectively, in paneis A and B). The absorption spectra in the Soret region of the ferrous native and carboxymethylated cytochrome c (dashed lines) are shown in panels A’ and B’, respectively. X-band EPR spectra and absorption spectra in the Soret region were obtained at pH 7.0, in 1.0 X 10-l M N-[2-~~~~~~~~~~~~~~~~-~‘~~~ L ~cha%%s&?kn~ a+‘N~&GH &L&k sq’tem prrpj 1.0 X 10-l M NaCl, and 100 K or 10°C respectively. X-band EPR spectra and absorption spectra in the Soret region were obtained at l.DX 1Dm3 M and 5.flX 30-’ M prorein concentration, respectively. Setting conditions for X-band EPR spectra were: 9.42 GHz microwave frequency; 20 mW microwave power; 0.10 mT modulation amplitude. For additional experimental details, see text.

276 P. Ascenzi et al.

Values of the equilibrium constant for the association of nitric oxide to ferrous native and carboxymethylated cytochrome c were determined at 10°C from absorbance changes accompanying ligand binding, between 400 nm and 440 nm (see Fig. 1, panels A’ and B’). In a typical experiment, a buffered solution of ferrous native or carboxymethylated cytochrome c (5.0 X 10m6 M) was introduced anaerobically into a l-cm spectrophotometric cell fitted with a serum cap for the injection of the nitric oxide solution; no gaseous phase was present. Various aliquots of the buffered solution (kept in a closed vessel under nitric oxide at 7.6 Torr, 76.0 Torr, or 760.0 Torr) were added to the ferrous native or carboxymethylated cytochrome c solution by using a precision microsy- ringe. After each addition, the system was equilibrated at 10°C requiring less than 30 min, and the spectrum recorded on a double-beam spectrophotometer [see also Ref. 141. The solubility of nitric oxide in the aqueous buffered solution at 10°C is 2.45 x 1O-3 M [see Ref. 151.

The reaction of ferrous native and carboxymethylated cytochrome c with nitric oxide is completely reversible, since spectra revert to the initial ones by merely pumping off the nitric oxide or bubbling nitrogen through the solution.

X-band EPR spectra of ferrous nitrosylated native and carboxymethylated cytochrome c were collected at 100 K on a BRUKER ESP 300 spectrometer.

Absorption spectra of ferrous and ferric forms of native and carboxymeth- ylated cytochrome c were recorded at 10°C on a JASCO J-510 and a VARIAN Cary 219 double-beam spectrophotometers.

All the data were obtained at pH 7.0, in 1.0 x lo--’ M N-[2-hydroxyethyl] piperazine-N’-[2_ethanesulfonic acid]/NaOH buffer system plus 1.0 x lo- ’ M NaCl.

RESULTS AND DISCUSSION

The X-band EPR spectra and the absorption spectra in the Soret region of the nitric oxide derivative of ferrous native and carbovethylated cytochrome c, at pH 7.0 and 100 K or lo”C, are shown in Figure 1. X-band EPR spectra of the nitric oxide derivative of ferrous native cytochrome c shown in Figure 1 can be superimposed on those obtained in solution and in the powdered form [6, 71. The direct binding of nitric oxide to the heme iron of ferrous native and carboxymethylated cytochrome c through the nitrogen atom is evidenced by the triplet of triplets and the doublet of triplets observed in the g, (= 2.008) region of the X-band EPR spectra shown in Figure 1 (panels A and B), obtained in the presence of r4N0 and 15N0, respectively [see also Refs. 6, 71.

EPR and absorption spectra of the nitrosylated derivative of ferrous native and carboxymethylated cytochrome c are similar. On the other hand, the absorption spectra in the Soret region of the ferrous native and carboxymeth- ylated cytochrome c display different molar absorption coefficients (131.2 mM-’ cm-’ versus 106.0 mM- ’ cm-‘, at 415 nm) (see Fig. 1, panels A’ and B’). This result probably reflects the hexa- and penta-coordination state of the heme-iron in the ferrous native and carboxymethylated cytochrome c, at pH 7.0 [see Refs. 2, 131.

The X-band EPR spectra of the nitric oxide derivative of ferrous native and carboxymethylated cytochrome c display a rhombic shape with a good resolution of-the nine-line (14NO) or the six-line (15NO) superhypertine structure in the g,

NITRIC OXIDE BINDING TO CYTOCHROME c 277

( = 2.008) region [see Refs. 6,7 and Fig. 1, panels A and B, respectively]. A good resolution of the superhypetfine structure in the g, region has also been reported to occur in the nitric oxide derivative of ferrous beef heart cytochrome a3 in cytochrome c oxidase [7]; horseradish and baker’s yeast cytochrome c peroxidase [12, 16, 171; A& sia limacina and elephant myoglobin [18, 191; Arenicola marina, Chironomous thummi thummi, and Octolasium complanatum erythrocruorin [20-221; and Scupharca inaequivalvis tetrameric hemoglobin [23]. On the other hand, the X-band EPR spectra of the nitrosylated derivative of ferrous Cotyphaena hipputus, Dermochelys coriacea, horse and sperm whale myoglobin [20], Dicrocoelium dendriticum monomeric hemoglobin [24], Scupharcu inaequivalvis dimeric hemoglobin [23], camel, horse, and human tetrameric hemoglobin in the high affinity state [25-311 display a weak superhyperfine structure in the g, region. The different degrees of resolution of the superhyper- fine structure in the g, region as observed in different hemoproteins have been tentatively related to the degrees of interaction of the unpaired electron spin density with the iron bound proximal histidine NE2 atom [12, 17, 20, 26, 321.

Beside the g, region analyzed thus far, the hyperfine splitting in the low magnetic field region (g, = 2.07) of the X-band EPR spectrum of the nitric oxide derivative of ferrous native and carboxymethylated cytochrome c [see Refs. 6, 7 and Fig. 1, panels A and B] is similar to that of the corresponding derivative of beef heart cytochrome a3 in cytochrome c oxidase 171; horseradish and baker’s yeast cytochrome c peroxidase [12, 16, 171; Aplysia limacina and elephant myoglobin [18, 191; Arenicola marina, Chironomus thummi thummi, and Octo- lasium complanatum erythrocruorin [20-221; and Scapharca inaequivalvis tetra- meric hemoglobin [23], but differs significantly from that of Coryphaena hipputus, Dermochetys coriucea, horse and sperm whale myoglobin [20], Dicrocoelium dendriticum monomeric hemoglobin [24], Scapharca inaequivalvis dimeric hemo- globin [23], camel, horse, and human tetrameric hemoglobin in the high affinity state [25-311. As suggested by John and Waterman [33], such a finding may reflect different stabilization modes of the iron-bound nitric oxide molecule in the distal site of the heme pocket. However, in the absence of comparable structural information no further conclusion can be drawn on the origin of this spectroscopic difference.

Differences in the g, and g, region of the X-band EPR spectra of the ferrous nitrosylated hemoproteins considered are paralleled by variations also of the absorption spectra, in the Soret region. Thus, around neutrality, the molar absorption coefficient in the Soret region of the nitrosylated derivative of ferrous native and carboxymethylated cytochrome c, horseradish and baker’s yeast cytochrome c peroxidase, Aplysiu limacina myoglobin, Scapharca inaequiv- alvis dimeric hemoglobin, and camel, horse, and human tetrameric hemoglobin, in the high affinity state ranges between 90 mM_’ cm-’ and 150 mM_’ cm-‘, the value of h,,, changing from 416 nm to 420 nm [see Refs. 12, 17, 18, 25, 27-31, 34 and Fig. 1, panels A’ and B’].

Figure 2 shows the ligand binding isotherms for the association of nitric oxide to ferrous native and carboxymethylated cytochrome c, at pH 7.0 and 10°C. Applying the minimum model accounting for an apparent single binding site per heme-iron (i.e., per molecule), a relation between the nitric oxide equilibrium dissociation constant for ferrous native and carboxymethylated cytochrome c (K) and the molar fraction of the ligand-bound nitrosylated hemoprotein (y),

278 P. Ascenzi et al.

I I I

0 2 4 6 8 10

L-x01 / PO1 FIGURE 2. Ligand binding isotherms for nitric oxide association to ferrous native (0) and carboxymethylated (0) cytochrome c. The arrow indicates the endpoint approximat- ing P = 1 at [X,] 2 1OO.K at a constant protein concentration. The concentration of nitric oxide (i.e., [X,]; M) is that of the total ligand [see Ref. 351. The protein concentration (i.e., [P,]; M) is 5.0 x 10e6 M. The continuous lines were generated from Eq. (1) with the following parameters: K/[P,] = 1.64 and K= 8.2 X 10m6 M for native cytochrome c; and K/[P,] = 0.01 and K s 5 X 10--s M for carboxymethylated cytochrome c [see Ref. 351. Values of K were obtained with an iterative nonlinear least-squares curve fitting procedure. The data were obtained at pH 7.0, in 1.0 X 10-r M N-[2-hydro~ethyl]piperazine-N’-[2-ethanesulphonic acid]/NaOH buffer system plus 1.0 X 10-l M NaCI, and 10°C. For additional experimental details, see text.

under conditions where the total protein concentration [PJ is not negligible with respect to K, may be expressed according to Eq. (1) [3.5]:

&I =y+ K y

IP,l Ip,1’c-T (1)

where [X, 1 indicates the total ligand concentration. Equation (1) allows evalua- tion of values of K, under conditions where the concentration of the bound ligand is not negligible with respect to that of the total ligand (i.e., under conditions where 0.1. K I [P,] I 100. K) [35]. In fact, for [P,] < 0.1. K, the con- centration of the bound ligand is negligible with respect to that of the total ligand, the latest approximating that of the free ligand from which the value of K is determined [35]. On the other hand, for [PO] > 100-K, the concentration of the bound ligand approximates that of the total ligand, the free ligand concen- tration being negligible; thus, values of K cannot be determined but only taken as maximum estimates [35]. Therefore, under these conditions, the estimate of K depends also on the protein concentration. The minimum protein concentration which has been used in the present study ([P,,] = 5.0 x 10M6 M) depends on the minimum detectable amplitude of the absorbance change(s) accompanying the hemoprotein nitrosylation (A E = 15.5 mM_’ cm-’ at 417.0 nm, and 16.2 mM_’ cm-’ at 413.7 nm for ferrous native and carboxymethylated cytochrome c, respectively) (see Fig. 1, panels A’ and BY, using a l-cm path length cuvette.

Equation (11, which describes the physical quantity y as a function of the total nitric oxide concentration at a given protein concentration (i.e., [X,]/[P,,], where [Pal = 5.0 X 10e6 M) has been used to generate the continuous lines

NITRIC OXIDE BINDING TO CYTOCHROME c 279

shown in Figure 2; the agreement with the experimental data is fully satisfactory giving us confidence on the correct assumption(s) underlying Eq. (1) (see Fig. 2). From the dependence of y versus [X,]/[P,] (see Fig. 2), values of K for nitric oxide binding to ferrous native and carboxymethylated cytochrome c (= 8.2 X lO-‘j M and I 5.0 x 10e8 M, respectively), were obtained. According to Eq. (11, the value of K for nitric oxide binding to ferrous carboxymethylated cytochrome c can be taken only as a maximum estimate since, whenever [PJ > 100. K, the addition of 0 to 1 mole of ligand per equivalent of protein gives complete binding of the ligand within the limits of the experimental error and [X,l/[P,l = 1 [351.

The affinity of nitric oxide for ferrous carboxymethylated cytochrome c (K I 5.0 X 10m8 M) is higher by at least two orders of magnitude than that observed for ligand binding to the ferrous native form (K = 8.2 X lop6 M), reflecting the loss of free energy associated to the replacement of the methionyl residue at position 80 by nitric oxide [6, 71. In this respect, the affinity of nitric oxide for ferrous carbovmethylated cytochrome c (K I 5.0 X 10m8 M) approxi- mates that estimated for nitrosylation of sperm whale myoglobin as well as of human hemoglobin (K = 2.7 X lo- ” M and 5.8 x lo-l3 M, respectively, at pH 7.0 and 20°C) [36], both showing a ferrous ligand free penta-coordinated heme iron [see Ref. 371. Next, from values of K for nitric oxide binding to ferrous native and carboxymethylated cytochrome c a minimum free energy value of 10 kJ mol-‘, at lO”C, may be estimated for the heme-iron Met80 bond.

As a whole, present results (i) give a detailed characterization of nitric oxide binding to ferrous native and carboqmethylated cytochrome c, (ii> show clearcut evidence for the diatomic gaseous ligand-induced cleavage of the Fe-Met80 bond in ferrous native cytochrome c, and (iii) allow estimation of the free energy associated to the heme-iron sixth coordination bond ( > 10 kJ mall’, at 10°C).

Authors thank Professor M. Brunori (Department of Biochemical Sciences “Alessandro Rossi Fanelli, ” University of Rome ‘La Sapienza, “Rome Italy), Professor G. Rotilio (Department of Biology, University of Rome “Tor Vergata, ” Rome, Italy), Professor A. Schejter (Depart- ment of Biochemistry, University of Tel Aviv, Israel), and Professor M. T. Wilson (Department of Chemistty and Biological Chemistry, University of Essex, Colchester, United Kingdom) for helpful discussions. This work was supported by grants of the Italian Ministry for University Scientific Research and Technology (Minister0 per I’Universitci e la Ricerca Scientifica e Tecnologica), as well as of the Italian National Research Council (Consiglio Nazionale delle Ricerche; target oriented project: Chimica Fine ZZ).

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Received January 21, 199.3; accepted March 30, 1993