Eur. J. Biochem. 204, 107-112 (1992)
(0FEBS 1992
NOE and two-dimensional correlated H-NMR spectroscopy of cytochrome c ' from Chromatiurn vinosum Lucia BANCI, Ivano BERTINI, Paola TURANO and Margarita VICENS OLIVER Department of Chemistry, University of Florence, Italy (Received July 29/0ctober 29, 1991) - EJB 91 1003
'H two-dimensional (nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) spectra of cytochrome c' from Chromatiurn vinosum have been obtained. The protein is of medium size (MI 28000), essentially high spin (5' = 5/2) although some quantum mechanical spin admixing with S = 3/2 may be present. Under these circumstances NOESY cross peaks have been revealed between geminal protons (a-CH2 propionate and fl-CH2 protons of the bound histidine) and between a-CH2 propionate protons and the heme methyl groups. COSY maps have confirmed the geminal nature of the proton pairs, even with a linewidth as large as 900 Hz; the J value is about 12 Hz. This assignment has rationalized on a sound basis the biochemical behavior of this protein with pH and has showed the utility of this kind of spectroscopy for the other cytochromes c' structures and analogous systems.
Two-dimensional (2D) NMR experiments have been shown to be quite meaningful also in the case of paramagnetic metalloproteins, where large longitudinal relaxation rates and linewidths may be operative [l]. Besides the P-CH2 protons of cysteines bound to iron in some Fe - S proteins [2, 31, 2D NMR spectra have been mainly recorded on low-spin iron(II1) heme proteins 14 - 111. The latter proteins are characterized by large electron relaxation rates and therefore by relatively small nuclear relaxation rates. The combined use of COSY and NOESY experiments has lead to the assignment of proton signals from groups attached to the heme moiety (vinyl and propionate side chains), to establish their orientation with respect to the heme methyl groups and sometimes to assign the signals of the heme methyl groups as well as signals close to but not bound to the iron(II1) ion. On the contrary, highspin iron(II1) is characterized by slow electron relaxation rates and therefore induces fast nuclear relaxation rates and large linewidths, making the 2D-NMR characterization much more difficult. Indeed, up to now, only one report is available on high-spin iron(II1) systems [12]. Cytochrome c' species are a class of proteins present in a variety of photosynthetic and denitrifying bacteria. They are proposed to be involved in electron-transfer processes [I 31. The X-ray structure is available at high resolution for Rhodospirillum molischianum [14] and a preliminary X-ray study for the C. vinosum has appeared [15]. Cytochrome c' from C. vinosum is a protein of medium size (Mr 28000) constituted by two identical subunits, each of them containing a heme moiety covalently bound to two cysteinyl residues through thioether links (Fig. 1) 116,171. The Correspondence to I. Bertini, Department of Chemistry, University of Florencc, Via Gino Capponi, 7, 1-50121 Florence, Italy Abbveviutions. 2D, two dimensional; NOESY, nuclear Overhauser effect spectroscopy; COSY, two-dimensional correlated spectrosCOPY.
cys
cys-x-Y-
I
I
1
i
H3CCH /
i
-CHCH,
N'
N ' .CH3
"'i i"'
HOOC
COOH
Fig. 1. Schematic drawing of the heme present in cytochromes c'.
iron is in the 3 + oxidation state and is coordinated by an axial histidine as the fifth ligand [18]. In a variety of five-coordinated [19, 201 and six-coordinated [21] (in the latter case with very weak axial ligands) iron(II1) heme compounds, the ground spin state has been proposed to be a quantomechanical spin admixture of S = 5/ 2 and 5' = 312 spin states on the basis of EPR, NMR and magnetic measurements. This spin-admixed state has been proposed also for C. vinosum cytochrome c' at neutral pH [22,
108
a
80
60
40
20
0
G(PPn-4
,
I 80
I
I
I
60
40
20
I 0
6 (ppm) Fig. 2. 600-MHz 300-K 'H-NMR spectra of C. vinosum cytochrome c' at p H 4.5 (a) and 8.5 (b). Both spectra were rccorded in D20solution.
231, despite the magnitude of the proton hyperfine shifts of the heme methyl groups is essentially consistent with a highspin iron(II1) ion [24-261. This class of proteins shows a remarkable pH dependence of the spectroscopic properties as detected by a variety of spectroscopic techniques (visible, EPR, Mossbauer, magnetic circular dichroism, resonance Raman and NMR) [23, 27341. The changes with pH have been interpreted on the basis of several pH-modulated equilibria [23, 31 - 341. One pK, (of about 6) has been proposed to originate from the deprotonation of a heme propionate side chain while the second one
(pK, in the range 8 - 9) would monitor the deprotonation of the iron-bound histidine [35] (with the contemporary change of the spin state to a pure high spin S = 512) [22, 36, 371. We have applied 2D-NMR techniques to this system with the aim of the following: (a) extending the applicability of 2D NMR to high-spin iron(II1) systems which display very broad lines; (b) assigning on a firm basis the spectra and therefore gaining a better understanding of the conformational variation which have been proposed to occur on the basis of monodimensional NMR as well as of the other spectroscopic techniques 123, 31 - 341.
109 I
I
I
80
.
netic systems, where usually T2
(0FEBS 1992
NOE and two-dimensional correlated H-NMR spectroscopy of cytochrome c ' from Chromatiurn vinosum Lucia BANCI, Ivano BERTINI, Paola TURANO and Margarita VICENS OLIVER Department of Chemistry, University of Florence, Italy (Received July 29/0ctober 29, 1991) - EJB 91 1003
'H two-dimensional (nuclear Overhauser effect spectroscopy (NOESY) and two-dimensional correlated spectroscopy (COSY) spectra of cytochrome c' from Chromatiurn vinosum have been obtained. The protein is of medium size (MI 28000), essentially high spin (5' = 5/2) although some quantum mechanical spin admixing with S = 3/2 may be present. Under these circumstances NOESY cross peaks have been revealed between geminal protons (a-CH2 propionate and fl-CH2 protons of the bound histidine) and between a-CH2 propionate protons and the heme methyl groups. COSY maps have confirmed the geminal nature of the proton pairs, even with a linewidth as large as 900 Hz; the J value is about 12 Hz. This assignment has rationalized on a sound basis the biochemical behavior of this protein with pH and has showed the utility of this kind of spectroscopy for the other cytochromes c' structures and analogous systems.
Two-dimensional (2D) NMR experiments have been shown to be quite meaningful also in the case of paramagnetic metalloproteins, where large longitudinal relaxation rates and linewidths may be operative [l]. Besides the P-CH2 protons of cysteines bound to iron in some Fe - S proteins [2, 31, 2D NMR spectra have been mainly recorded on low-spin iron(II1) heme proteins 14 - 111. The latter proteins are characterized by large electron relaxation rates and therefore by relatively small nuclear relaxation rates. The combined use of COSY and NOESY experiments has lead to the assignment of proton signals from groups attached to the heme moiety (vinyl and propionate side chains), to establish their orientation with respect to the heme methyl groups and sometimes to assign the signals of the heme methyl groups as well as signals close to but not bound to the iron(II1) ion. On the contrary, highspin iron(II1) is characterized by slow electron relaxation rates and therefore induces fast nuclear relaxation rates and large linewidths, making the 2D-NMR characterization much more difficult. Indeed, up to now, only one report is available on high-spin iron(II1) systems [12]. Cytochrome c' species are a class of proteins present in a variety of photosynthetic and denitrifying bacteria. They are proposed to be involved in electron-transfer processes [I 31. The X-ray structure is available at high resolution for Rhodospirillum molischianum [14] and a preliminary X-ray study for the C. vinosum has appeared [15]. Cytochrome c' from C. vinosum is a protein of medium size (Mr 28000) constituted by two identical subunits, each of them containing a heme moiety covalently bound to two cysteinyl residues through thioether links (Fig. 1) 116,171. The Correspondence to I. Bertini, Department of Chemistry, University of Florencc, Via Gino Capponi, 7, 1-50121 Florence, Italy Abbveviutions. 2D, two dimensional; NOESY, nuclear Overhauser effect spectroscopy; COSY, two-dimensional correlated spectrosCOPY.
cys
cys-x-Y-
I
I
1
i
H3CCH /
i
-CHCH,
N'
N ' .CH3
"'i i"'
HOOC
COOH
Fig. 1. Schematic drawing of the heme present in cytochromes c'.
iron is in the 3 + oxidation state and is coordinated by an axial histidine as the fifth ligand [18]. In a variety of five-coordinated [19, 201 and six-coordinated [21] (in the latter case with very weak axial ligands) iron(II1) heme compounds, the ground spin state has been proposed to be a quantomechanical spin admixture of S = 5/ 2 and 5' = 312 spin states on the basis of EPR, NMR and magnetic measurements. This spin-admixed state has been proposed also for C. vinosum cytochrome c' at neutral pH [22,
108
a
80
60
40
20
0
G(PPn-4
,
I 80
I
I
I
60
40
20
I 0
6 (ppm) Fig. 2. 600-MHz 300-K 'H-NMR spectra of C. vinosum cytochrome c' at p H 4.5 (a) and 8.5 (b). Both spectra were rccorded in D20solution.
231, despite the magnitude of the proton hyperfine shifts of the heme methyl groups is essentially consistent with a highspin iron(II1) ion [24-261. This class of proteins shows a remarkable pH dependence of the spectroscopic properties as detected by a variety of spectroscopic techniques (visible, EPR, Mossbauer, magnetic circular dichroism, resonance Raman and NMR) [23, 27341. The changes with pH have been interpreted on the basis of several pH-modulated equilibria [23, 31 - 341. One pK, (of about 6) has been proposed to originate from the deprotonation of a heme propionate side chain while the second one
(pK, in the range 8 - 9) would monitor the deprotonation of the iron-bound histidine [35] (with the contemporary change of the spin state to a pure high spin S = 512) [22, 36, 371. We have applied 2D-NMR techniques to this system with the aim of the following: (a) extending the applicability of 2D NMR to high-spin iron(II1) systems which display very broad lines; (b) assigning on a firm basis the spectra and therefore gaining a better understanding of the conformational variation which have been proposed to occur on the basis of monodimensional NMR as well as of the other spectroscopic techniques 123, 31 - 341.
109 I
I
I
80
.
netic systems, where usually T2
