Assignment of the redox potentials to the four haems in Desulfovibrio vulgaris cytochrome c3 by 2D-NMR

Volurae 314, number 2, 155-158

FEBS 11851

December 1992

© 1992 Federation of European Blochemtcal Societie~ 00145793/9~$5.00

Assignment of the redox potentials to the four harms in Desulfovibrio vulgaris cytochrome c3 by 2D-NMR C a r l o s A. Salgueiro", D a v i d L. T u r n e r b, H e l e n a S a n t o s ~, J e a n L e G a l l ~ a n d A n t 6 n i o V. Xavier" "Centre de Tecnologla Quirnica e Biol6gica and Umversidade Nova de L~sboa, Apt 127, 2780 Oeiras, Portugal, ~Department of Chemtstry, Ungversio, of Southampton, Southampton S 0 9 ~NH, UK and CDepartmettt of Oiochemistr),, UtffverMty of Georgia, Athens, Georgia 30602~ USA

Received 19 Augus~ 1992 accordins to their redox potential. Tile .~trate~y used was based on a complete network of chemical exchange connect~vities between the NMR s~gnals obtained for all oxidation levels to the corresponding ones m the fully reduced spectrum [1992, Eur. J. B~ochem., m press]. This unequivocal eross-ass~gnment disagrees w~th earlier results obtained for the simdar protein from De~ulJovlbrto vulgarls (M~yazaki F ) [1991, FEBS Lett. 285, 149-151]. ~-~jtoclirome c~, ~a'udfi~aem prote~h; _ r G - ~ ' R ' ; K'u'3:%~ 3alo'qz~W,A'-ray b~tructttre

1. I N T R O D U C T I O N Multiredox centre proteins are involved in several crucia'i steps or reactions in which f'undamenta~ eaergy transduction phenomena lake place. However, the complexity o f these proteins has hindered the determination o f the precise physico-chemical parameters necessary to explain the intricate mechanisms involved, the architecture of the different redox centres, and the indispensable cross-assignment between each of the redox centres within the structure and the measurable parameters. Cytoehrome c3, a tetrahaem protein isolated from Desulfovibrio spp. [1]~ being a small (13 kDa) soluble protein for which X-ray structures are available [2-4]° has been subjected to several physicoclaemical and spectroscopic studies [5-12]. Thus, a wealth o f information is available for several homologous proteins from different Desulfovibrio spp. and much effort has been dedicated to the cross-assignment o f the redox potentials o f the individual harms to their position in the 3D structure [8-15]. During a redox titration, the 16 possible combinations of redox states o f the individual harms can be grouped in a series of five macroscopic oxidation states Correspondence addresa. A.V. Xavier, Centre de Tecnolo~m Quhnica e Bi016~ica, Apt. 127, 2780 Oeiras, Portugal. Fax: (351)(1)4428766. Abbrev~attons. c3DvMF, Deau6¢ovtbrto vulgarts (Mlyazakl F.) eyto-'hrome c~; c3Dvlt, De~ulfovlbrto vu[garts (Hildenborough) cytochrome c~; ¢3DbN, DesulfovJbriobaculatus (Norway) eytoehrome c3; ROESY, Rotatin 8 Frame N e E Spt:ctro~:opy, NOESY, N e E Spectroscopy; Ml, harm methyl groups 0 = 2 t, 7 j, 12 ), 18 L) according to the IUPAC-IUB nomenclature.

Publtshed by Elsevier Science Publishers B V.

[7], which we shall refer to as stages, and which are connected by four one-electron steps. Each o f the five stages (numbered from 0 to 4, according to the number of oxidised harms) comprises a number or intermediate oxidation states, each having the same n u m b e r of oxidised h a r m s [7]. Since the h a r m iron atoms are in the low-spin state they are diamagnetic in the reduced form and paramagnetic when oxidised. Thus, N M R spectroscopy has been shown to be a useful technique to follow the redox stages o f this protein as well as to measure the degree o f oxidation o f each harms, which have.di,q~rent redox potentials (individual microscopic redox potentials) at every stage [7,9]. As the interconversion between oxidation states belonging to the same stage (intramolecular electron exchange) is fast on the N M R time scale and the interconversion between states belonging to different stages (#)termolecular exchange) is slow, a separate set o f signals is observed for the protons o f the harms in each different stage. Indeed, under these conditions the distribution of paramagnetic shifts observed for each stage is governed by the relative microscopic redox potentials [7,8]. The comparatively simple N M R spectra o f the fully reduced or fully oxidised protein provide suitable starting points for assignment. Earlier work [8,9] has concentrated on the fully oxidised form (stage 4), but few specific assignments could be obtained. We have used 2 D - I H - N M R to assign all o f the h a e m methyl groups of ferrocytochrome c3 (stage 0 ) unequivocally to individual h a r m s in the three dimensional structure [16]. Since 2 D - 1 H - N M R techniques carl also be used to detect exchange processes between redox stages of cytocgrome c3 [17] it is then possible to follow each haem 155

Volume 314, r.umber 2

FEBS L E T T E R S

F1

December 1992

2°~ 1°

ppm

s

|

lO

A 15

2;

3= 20

2a~ ' i 4

25

ao

~4 4 25

30

20

15

10

F2 p p m

Fig. 1 - Combined I'qOESY (A) and ROESY (B) contour plots for the different levels of oxidation in c3DvH at 298 K and p-'H=8.9. The spectra were obtained in a 500 MHz Braker AMX-500 ~pectrometer flom a 2 mM solution of c3DvH previously lyophilized and resuspended in :H:O [16], The full), reduced stage was achieved by addition or'catalytic amounts of D. gtgas hydrogenase ila a hydrogen atmosphere [16], The intermedmte oxidatmn levels were obtained by fir.~t washing out the hydrogen from the redltced sample with mtrogen and then adding controlled amounts of mr into th~ NMR tube w~th a Hamilton syringe through serum caps. The NOESY spectrum was collected (512 x 2048 data ~ize) with a mixm8 time of 25 ma and tranfformed with a Gaus~ian apodi~ation (Ib = -15 I-Is, gb = 0,037) in Fn and a shifted ~ine bell mulhplieation (cosine) i,1 F~ with zero filhng to 1,024 data points in F t. la the ROESY spectrum the same type of transformation was used (lb = -15 Hz, gb=0.045) and the pulse for spin leek was 10 ms. Chemical shifts are relahve to 3-ttr~methyhilyl) propane sullbmc acid (sodium ~alt), The NOESY spectrum emphasizes the first three ztages of oxidation (0--3)and the ROESY ~pectrum ~hows the last ~tep ofoxidatmn (~tage 3-4), Cross peaks which conner the chemical shifts of.~el~cted methyl groups in stages 0-4 to their shift~ in stage 3 have been labelled. The numberb (1--4) identify the haems according to the redox potential (starting from the lowest one, thus corl'espondm8 to haems 11I, II, I, and IV, respectively). The superscripts (0-4) indicate the partLealar stage Thus, the shifts of the~e group~ (ef Table 1) should be read from the left hand (FI) scale, Negative peaks are indicated with dotted hnes. 1D spectra of the samples are plotted at the ed3c of the corresponding hall-spectra,

m e t h y l g r o u p t h r o u g h o u t every o x i d a t i o n stage. T h i s s t r a t e g y is used here t o o b t a i n t h e full c r o s s - a s s i g n m e n t o f the f o u r h a e m s within the X - r a y s t r u c t u r e a c c o r d i n g t o their r e d o x p o t e n t i a l . 2. R E S U L T S

AND DISCUSSION

Fig. 1 s h o w s the N O E S Y (A) a n d R O E S Y (B) s p e c t r a o f e 3 D v I - I s a m p l e s (see e x p e r i m e n t a l c o n d i t i o n s in the figure legend) p o i s e d a t i n t e r m e d i a t e o x i d a t i o n levels f o r w h i c h p o p u l a t i o n s belougi~ag t o ~-edox stages 0 - 3 (Fig. 1A) a n d 3--4 (Fig. i B) co-exist. T h e signals f o r e a c h h a e m m e t h y l g r o u p at individual o x i d a t i o n stages give rise t o a u n i q u e n e t w o r k o f c r o s s - p e a k s due to i n t e r m o l 156

e c u l a r e l e c t r o n t r a n s f e r steps ( T a b l e I). T h e cross-ass i g n m e n t o f the p a r a m a g n e t i c a l l y shifted r e s o n a n c e s (stages 1-4) to those due to the fully r e d u c e d stage 0 [16] allows a n u n e q u i v o c a l c r o s s - a s s i g n m e n t t o each h a e m in the t h r e e - d i m e n s i o n a l s t r u c t u r e (see T a b l e I). R O E S Y s p e c t r a [18] were used t h r o u g h o u t the r e d o x t i t r a t i o n in o r d e r to differentiate cross p e a k s due t o c h e m i c a l exc h a n g e (positive intensities) f r o m those d u e to N O E ' s ( n e g a t i v e intensities)° which a r e b o t h p o s i t i v e in th~ N O E S Y spectra. T a b l e I also ~.ow-~t" ..... tlle l~l t.' .l ..~.l , / U: l.l _ IkJl _e 4_..:a~.:^~ ~O.] l w. ,.l l.. . .l ~.l , A ,.,#Al~gt.l~Jll O f o r e a c h o f the f o u r h a e m s in e a c h stage, c a l c u l a t e d f r o m the r e l e v a n t chemical shifts o b s e r v e d f o r o n e m e t h y l g r o u p c h o s e n f o r each h a e m . T h i s c a l c u l a t i o n is simply

Volume 314, number 2

FEBS LETTERS

December 1992

Table I Chemical shift~ of one methyl group for each haem m the five oxldatton stages and corresponding oxldat~on fractions, x, Stage

0 1 2 3 4

Chemical shifts L0pm)

z,

~'x,

NI12~

M7~

MIS~~

MlS~v

MI2~

M7~

MIS?

MIS,½

3.41 15 61 13.98 18.96 20.71

3.13 4.93 16.10 21.54 22.00

3.27 5.72 17.20 28.57 29.69

3.26 6.09 7.01 8.20 30.13

00 0.705 0.611 0.899 1.0

0.0 0.095 0.687 0.975 10

0.0 0.093 0.527 0.957 10

(}.0 0.105 0.139 0 183 1.0

based on the ratio of the paramagnetic shifts of the methyl groups at each stage to their total shifts in the fully oxidised protein [7,8]. In order to minimize errors arising from extrinsic paramagnetic shifts (i.e. dipolar shifts caused in the proton signals of one haem by any of the other baems [7]) the haem methyl groups chosen to probe the degree of oxidation of each haem in the successive oxidation stages and depicted in Fig. 1, were those of groups pointing towards the protein surface and therefore far from the other haems: MI2]H, M7]l, M181 and Ml81v (Roman numbers indicating their position in the primary sequence [19]). It is also assumed that the distribution of Fermi contact shifts does not vary between stages. Analysis of T,qble I shows that neither the extrinsic shifts nor any variation of the Fermi contact ones can contribute significantly since the sums of the oxidation fraction at each stage are very close to integers. 3. CONCLUSIONS Table I shows that haem III becomes largely oxidised in step 1 (stage 0-1), and is followed by haems II and I which both oxidise to a similar extent in steps 2 and 3, and finally by haem IV in step 4. The fraction of oxidation for each step can also be used to calculate the relative mid-point redox potentials as well as the interacting potentials [7,8] of the haems at each stage (manuseript m preparation). Although the X-ray structure of c3DvH cytochrome e~ has recently been obtained [4] its coordinates are not yet available and so those o f c 3 D v M F [3] were used for the assignment of stage 0 [16]. The two proteins are closely homologous~ as is the relative architecture of their four haems0 which is also maintained in the much less homologous protein c3DbN [20]. Furthermore, comparison of the 1D-NMR spectra of c3DvMF [9] and c3DvH [6] shows that the redox patterns are quite simi!ar, though not identical, as indeed are the haem methyl group shifts for the fully oxidised state. A previous identification of the four haems of c3DvMF was attempted by 1D-1H-NMR using a few

0.0 0.998 1.964 3.014 4.0

specific assignments made in stage 4 [8-10]. The assignment obtained differs from oars in that the earlier work assigned the largest change in oxidation in the first step to haem II whereas we find that the major change for haem I1 occurs in step 2 and that it is haem Ill which becomes largely oxidised in step 1. The present work relies on an extensive network of chemical exchange patterns connecting all the redox stages. Comparison of the spectra shows that methyl D o f Ref. 8 which was assigned to haem lll is in fact M7h. The earlier assignment is unconvincing since it was made solely on the basis of the proportional change of the paramagnetic shift in the first reduction step which is comparable [8] with that of the firmly assigned M2ht [8-10]. Indeed, the magnitude of the extrinsic shifts may be such that a comparison of the proportional changes in a single step (el. Table I) could be quite misleading, particularly in this case, since M2]a gives a strong NOE (i.e. it is very close) to a methyl group which belongs to the haem most reduced in that same step, M 121v [8-10]. There may be small differences in the relative redox potentials of the four haems in the Miyazaki and Hildenborough proteins, but the overall pattern appears to be the same. However, a different order of redox potentials has been proposed for the much less homologous c3DbN on the basis of single crystal EPR studies [14]. Such a difference would reflect the influence of the polypeptide chain. Studies to elucidate the physicochemical and physiological significance of the control of redox potentials are currently in progress. REFERENCES [1] LeGail, J and Fauque, O. (1988) m: Biology of Ana~robie Microorgamsms (Zehnder, A.J.B. Ed.) pp. 587-639, John Wdey, New York, USA.

[2] Pierrot, M., Haser, R., Frey. M, Payan, F. and Astier, J.-P. (1982} J Biol. Chem. 257, 14341-14348. [3] Higushi, Y., Kusonoki, 1Vi., Matsuura, Y., Yasuoka, N. and Kakudo, M. (1984) J. Mol. Biol. 172, 109-139. [41 Monmoto, Y., Tani, T., Okmaura, H., Higuchi, Y and Ynsuoka, N. (1991) J. Biochera. (Tokyo) i10, 532-540. [5] Dobson, C.M., Hoyle, N.J., GeFaldcs, C.F., Bruschi, M., LeGall, J. WrieR, P.E. and Williamb, R.I.P. (1974) Nature 249, 425-429.

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[6] Moura, J,LG., Santos, H., Moura. I,, L¢GalI, J., Moore G,R., Williams, R.J.P. and Xavter, A.V. (1982) Ear. J. Bioohem. 127, 151-155. [71 Santos, H., Moura, J.J.O., Mourn, I., LeGail, J. and Xa~der, A.V. (1984) Eur. J. lltochem. 141,283-296. [81 Fan, K., Akatsu, H., Kyogukth Y. and Niki, K. (1990) Biochemistry 29, 2257-2263. [91 Park, J.-$., Kano, K., Niki, K. and Akutsu, H. (1991) FEllS Lett. 285, 149-151. [10] Park, J,-$., Kano, K., Moriraoto, Y., Higuehi, Y., Yasuoka, N., Osata, M., N,kh K. and Akatsu, H. (1991) J. Btomol. NMR 1, 271-282. [11] Benosman, H., As~o, M., Bertrand, P., Y~gl, T. and Ga),da, J -P. (1989) Eur. J. Blochem. 182, 51-5S. [191 Moara, I., T¢is¢ira, M , Hu2cnh, B.FI., LeGalI, J. and Moura, J J G. (1988) Ear J. Bloehern. 176. 365-369.

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[13] Della, A., Cambillau, C., Bianeo, P., Haladjian, J. and Bruschi, M. (1987) Biochem. B~ophys. Res. Commun. 147, 818-823. [14] Guigliardh, B., Bertrand, P., More, C., Haser, R. and Gayda, J.-P. (1990) J. Mol. Biol 216, 161-166. [15] Colotta, M., Catarino, T., LeGalI, J. and Xavier, A.V. (1991) Eur. J. Bioehem. 202, 1101-1106. [16] Turner, D.L., Salgueiro, C.A., LeGall, J. and Xawer, A.V. (1992) Ear. J. Diochcm. (in press). [17] Santos, H., Turner, D.L., L~GalI, J. and Xavier, A.'V. (1984) J. Magn. Reson. 59, 177-180 [18] Bothner-By, A . A , Stephens, R.L., Lee, J., Warren, C.D. and Jeanloz, R.W., (1984) J. Am. Chem. So¢. 106, 811-813. [19] Mathew~, F.S. (1985) Prog. Bioph),s. M01¢¢ Blol. 45, 1-56. [20] Coutinho, I.B., Turner, D.L., LeGail, J. and Xavier, A.V. (1992) Ear. J. Bioehem. 209, 329-333.