Vol. 14, No. 1-4 159 USE OF NMR RELAXATION MEASUREMENTS TO DERIVE THE BINDING SITE OF PLASTOCYANIN IN COMPLEXES WITH CYTOCHROME-F AND C Sandeep Modi 1 , Ewen McLaughlin 1 , Derek S. Bendall 1 , S. He 2 and J.C. Gray 2 Departments of Biochemistry 1 and Plant Sciences 2 University of Cambridge, Cambridge, CB2 1QW England (U.K.) 1 Introduction Plastocyanin (PC) is a small (M r 10 500), 'blue' copper protein which transfers electrons from cyto- chrome / to the primary electron donor of photosystem I (P700) in the photosynthetic electron trans- port chain. It reacts rapidly with cytochrome / in vitro and also with several other cytochromes, although somewhat more slowly [1].. The crystal structures of both oxidized and reduced poplar plastocyanin have been determin- ed and show that redox changes cause only small changes at the Cu site, leaving the structure of the rest of the molecule essentially unchanged [2,3]. The Cu atom and its ligands (His-37, Cys-84, His-87 and Met-92) are located in a hydrophobic pocket near one end of the molecule (the "northern" end) such that only the imidazole ring of His-87 (the northern histidine) is accessible to solvent (Fig 1). Studies with small molecules, such as [Fe(CN)6] 3 " and [Co(phen)3]3+, have identified two reaction sites on plastocyanin; one close to the copper ligand His-87 at the northern hydrophobic patch and the other close to the more remote Tyr-83 at the eastern acidic patch [4-8]. Chemical modi- fication of the acidic amino acid residues, inhibitory effects of small molecules and ionic strength
Transcript
Vol. 14, No. 1-4 159
USE OF NMR RELAXATIONMEASUREMENTS TO DERIVE THEBINDING SITE OF PLASTOCYANIN
IN COMPLEXES WITHCYTOCHROME-F AND C
Sandeep Modi1, Ewen McLaughlin1, Derek S. Bendall1,S. He2 and J.C. Gray2
Departments of Biochemistry1 and Plant Sciences2
University of Cambridge, Cambridge, CB2 1QWEngland (U.K.)
1 IntroductionPlastocyanin (PC) is a small (Mr 10500), 'blue' copper protein whichtransfers electrons from cyto-chrome / to the primary electrondonor of photosystem I (P700) inthe photosynthetic electron trans-port chain. It reacts rapidly withcytochrome / in vitro and alsowith several other cytochromes,although somewhat more slowly[1].. The crystal structures of bothoxidized and reduced poplarplastocyanin have been determin-ed and show that redox changescause only small changes at the Cusite, leaving the structure of therest of the molecule essentiallyunchanged [2,3]. The Cu atom and
its ligands (His-37, Cys-84, His-87and Met-92) are located in ahydrophobic pocket near one endof the molecule (the "northern"end) such that only the imidazolering of His-87 (the northernhistidine) is accessible to solvent(Fig 1).
Studies with small molecules,such as [ F e ( C N ) 6 ] 3 " and[Co(phen)3]3+, have identified tworeaction sites on plastocyanin; oneclose to the copper ligand His-87at the northern hydrophobic patchand the other close to the moreremote Tyr-83 at the easternacidic patch [4-8]. Chemical modi-fication of the acidic amino acidresidues, inhibitory effects ofsmall molecules and ionic strength
160 Bulletin of Magnetic Resonance
effects on electron transferstrongly suggest that cytochrome /binds at the remote eastern site[9,10]. However the pathway ofelectron transfer from cytochrome/ to the copper site in plastocyanin
L12 H87
E60
C
Fig. 1 : Structure of Plastocyanin. The Culigands and the side-chains of Tyr83 and theresidues of the eastern acidic patch are shownhere with the coordinates of poplarplastocyanin from the Brookhaven Database,except for substitution of Glu45 (as in peaplastocyanin) for Ser45. ^-Strands areshaded.
is not clear. One possibility is thatcytochrome / binds to the negativecharges of the eastern acidic patchand donates electrons via atunneling pathway starting atTyr-83 [5,11]. However, the
distance from Tyr-83 to thecopper ion is approximately 12A,whereas at the northern site thecopper ion is only 6A from thesurface of the molecule [11].
The recent development ofexpression systems for the smallblue copper protein, plastocyanin[12-15], has provided a valuabletool for study of the moleculardetails of its interaction with itsnative reaction partners (cyto-chrome / and photosystem I in thephotosynthetic electron transportchain). To examine the pathway ofelectron transfer from cytochrome/ to plastocyanin we have alteredTyr-83 to Phe-83 and Leu-83 bysite-directed mutagenesis of thepea plastocyanin gene [13-14].Measurements of binding cons-tants and electron transfer ratesindicate, that Tyr-83 not onlyforms part of the main route ofelectron transfer from cytochrome/ to plastocyanin but is also invol-ved in binding to cytochrome / .
Nuclear magnetic resonancespectroscopy has been establishedas a very convenient and effectivetechnique for structural studies ofproteins. For haemproteins, thecharacteristic hyperfine-shiftedNMR spectrum of a paramagnetichaemprotein carries the signatureof the electronic and structuralproperties of the haem group.Measurements of spin - lattice
Vol. 14, No. 1-4 161
relaxation (Tj) and spin-spinrelaxation (T2) times providesuseful methods for thedetermination of dissociationconstants and distances of variousnuclei from the paramagneticcentre in a protein-proteincomplex. Relaxation measure-ments were carried out to getinformation about the relativedispositions of the two proteins(PC and cytochromes) in theircomplexes.
2 Assignment ofProton NMR reso-nances for peaPlastocyanin
Proton NMR measurements werecarried out on a Bruker AM 500-MHz FT-NMR spectrometer at300K in 50 raM phosphate buffer(pH 6.0). Proton chemical shiftswere referred to a proton signal ofdioxan as a reference at 3.74 ppm.Proton NMR resonances for pea PCwere assigned using 2D-NMRspectroscopy at 300K (pH = 6.0).The conformation of the Phe-83plastocyanin was examined by^-NMR. A ID spectrum in H2O,compared with that of the wild-type protein, clearly demonstratedthe disappearance of the amideresonance of Tyr-83 at 9.40 ppm,
and the appearance of a newresonance at 9.37 ppm which islikely to be that of Phe-83 [13],although positive identificationmust await further 2Dexperiments. Overall the closesimilarity between the spectra ofthe mutant and wild-type proteinsindicated that the replacement ofTyr-83 with Phe-83 had not led tomajor conformational changesthroughout the protein. Insuffi-cient protein of the Leu-83mutant was available for NMRanalysis, so its conformation wasexamined by CD between 190 and260 nm. The spectrum obtainedfor the oxidized protein wasessentially identical to that of thewild-type. The Phe-83 mutantprotein also gave a closely similarspectrum. These results confirmthat the three proteins hadidentical gross conformations.
3 Kineticssurements
Mea-
Electron transfer from reducedcytochrome to oxidized plasto-cyanin was monitored at 422 nmwith an Applied Photophysicsstopped-flow spectrophotometer(SF.17MV). The rate of binding ofplastocyanin and cytochrome wasmeasured by following the
162 Bulletin of Magnetic Resonance
increase in absorbance of oxidizedcytochrome at 410 nm in thestopped-flow spectrophotometer.K& was determined by takingadvantage of the increased absor-bance of the Soret band ofcytochrome on binding toplastocyanin
The results reported in Table I[13,14] demonstrate convincinglythat Tyr-83 of plastocyanin is partof the main tunneling pathway forthe electron between the haemrings of both cytochrome c andcytochrome / and the Cu atom ofplastocyanin. A leucine residue inthis position is much less effective
and it seems likely that thefacilitation of electron transfer bytyrosine or phenylalanine is dueto the aromatic nature of the ring,as has been proposed in otherproteins. A striking differencebetween the kinetics of reductionof plastocyanin by the twocytochromes is that the wild-typeprotein and the Phe-83 mutantbehave identically towardscytochrome c, but not towardscytochrome / . In the latter casethe rate of reduction of themutant protein is about seventimes slower, a difference that canbe ascribed entirely to weaker
Table IKinetic parameters for reduction of pea plastocyanin by cytochromes c and/
Plastocyanin
CytochromeWild typePhe-83Leu-83
k2 (x 10-6)(M-V1)
c as donor3.263.280.421
Cytochrome/as donorWild typePhe-83Leu-83
40.65.430.955
* A(M-l)
125312951260
98901270968
•*a(xlO-6)(MrV1)
20.022.721.7
43.55.861.27
*.a(xl0<(s-1)
16.017.517.2
4.404.611.31
*) k( (x 10-3)(s-1)
3.112.960.340
62584.0
Values are given as mean + standard deviation. Data for cytochrome/and c are from[13].and [14] respectively.
Vol. 14, No. 1-4 163
binding. We therefore predict thatwhen the structure of cytochrome/ becomes known it will reveal asurface residue in the region ofthe exposed haem edge which iscapable of hydrogen bonding tothe -OH of Tyr-83.
4 NMR relaxation(Ti) measurements
Protein concentrations weredetermined from the followingabsorption coefficients: reducedhorse heart cytochrome c, £550nm= 2.76 x 104 M-icm"1; reducedoil-seed rape cytochrome / ,£554nm = 2.6 x 104 M - i c m - l ;oxidised plastocyanin, £597nm =
4.7 x 103 M- lcm-1 . Proton NMRrelaxation measurements werecarried out on a Bruker AM 500-MHz FT NMR spectrometer at300K. The samples were in 0.01 Mphosphate buffer (containing 90raM NaCl) at pH 6.0 (volume, 0.4ml). Proton NMR spectra of Cd-PCwere obtained by accumulation ofabout 160 transients at 16K datapoints in quadrature mode. Tofacilitate relaxation measure-ments, a redox-inactive form ofplastocyanin was prepared, inwhich Cu was replaced by Cd. 2DNMR spectra of Cd-PC showed thatthe conformations of the two
proteins are essentially the same.For the relaxat ion timemeasurements, samples weretreated with Chelex 100 (Bio-Rad)to remove any traces of free metalions. To obtain the longitudinalrelaxation time ( T i o b s ) , theinversion recovery method with180^-T -90° pulse sequence wasused.
Longitudinal proton relaxationtimes of Cd-PC were measured inthe presence of variousconcentrations of cytochrome (corf) to find the binding constantsand the distances from variousprotons of Cd-PC to thecytochrome iron atom. Observedlongitudinal relaxation time( T l o b s ) o f Cd-PC protonresonances can be considered asthe sum of the relaxation rates ofthe bound and free Cd-PCfractions and is related to Kj), T\ 5and Tif, where Kj) is the apparentdissociation constant of theCyt/Cd-PC complex, Tjb is the Ti
164 Bulletin of Magnetic Resonance
of the Cyt/Cd-PC complex, and Tifis the Ti of the Cd-PC in theabsence of the cytochrome. KTJ andT i b f o r Cd-PC binding tocytochrome was obtained from theabove data. Kj) obtained from NMRrelaxation measurements agreedvery well with the value obtainedfrom optical spectroscopy (TableI).
6 Determination ofDistance using
Measurements
The Solomon and Bloembergenequations were used to determine thedistance (r) of individual protons ofbound Cd-PC from the ferric centreof cytochromes. The distances forthese protons were used to get therelative position and conformation ofPC with respect to the ferric ion ofcytochromes. Our initial results showthat the ferric ion of cytochrome isvery near to the Tyr-83 residue ofPC, which is consistent with ourkinetics studies. This work is still inprogress.
