Tryptophan radicals formed by iron/oxygen reaction with Escherichia ...

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Feb 28, 1994 - volume of each stopped-flow reactant was 70 pl/shot. Stopped-flow ... First derivative EPR spectrum at 77 K of the interme- diates after 6 s of ...
THE JOURNAL OF B l o ~ l c . 4 C ~H E M I ~ Y Vol. 269, No. 16, Issue of April 22, pp. 11699-11702, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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is oxidized and deprotonated. The radical is stabilized in a hydrophobic pocket, about 10A away from the nearestprotein surface (9, 10). We have studied the corresponding i n vitro "reconstitution" reaction with a mutant E. coli apoprotein R2, Y122F, lacking the essential tyrosine at position 122. In this reaction the normal iron centeris still formed upon addition of Fez+ and0, (8, 111,but thereduction equivalent provided by Tyr'" now has to be supplied from elsewhere. Several relatively stable paramag(Received for publication, February 9, 1994, and in revised form, February 28, 1994) netic transients are observed by EPR spectroscopy. By use of protein with incorporated deuterium-labeled tryptophan resiMargareta SahlinS, Giinter Lassmandll, dues we have found at least two species, which may be assigned Stephan PotschB, Agneta SlabyS, to oxidized tryptophan radicals. Britt-Marie SjobergS, and Astrid Graslundll** From the Wepartment of Molecular Biology and MATERIALSANDMETHODS Illepartment of Biophysics, Stockholm University, For overproduction of the E. coli protein R2 mutant we used the Arrhenius Laboratories, S-106 91 Stockholm, Sweden, strain MC1009 with the plasmids pGP1-2 and MK5, the latter a rethe §Max Delbriick Center of Molecular Medicine, combinant derivativeof pTZ18R containingthe mutant nrdB gene' (11). Robert-Rossle-Strasse 10, 0-13125 Berlin, Germany, Growing this strain in an iron-depleted medium (withor without deuand the W a x Volmer Institute of Biophysical and terated tryptophan) gives the apoprotein form of R2 (12). The protein Physical Chemistry, Technical University of Berlin, was purified as described earlier (13, 14). L-Tryptophan-indole-d,(98%) 0-10623 Berlin, Germany was obtained from Cambridge Isotope Laboratories Ltd. EPR spectra were recorded usingan ESP 300E Bruker spectrometer, The active state of the small subunit, protein R2, of ribonucleotide reductase is formed by the reaction of equipped witha cold fingerDewar flask forstudies at 77 K. For stoppedapoprotein with Fez+and 0,, whereby the diferric site flow studies at room temperature the EPR spectrometer was coupled with a commerciallyavailablestopped-flowEPR accessory specially and a stable phenoxy free radical on a tyrosyl residue designed for biological applications as described previously (15). The (Tyr'=) is formed. Thecorresponding reaction was stud- volume of each stopped-flow reactant was 70 pl/shot. ied in the mutant Y122F R2. It leads to a normal iron site, Stopped-flow light absorption studies were performed using a DX yr'"now has to be 17MV Biosequential stopped-flow ASVD spectrofluorimeter from Apbut the reduction equivalentfrom T supplied from elsewhere. EPR spectroscopy shows for- plied Photophysics. mation of severalparamagnetic species on different RESULTSANDDISCUSSION time scales. Using apoprotein with deuterium-labeled tryptophan residues, at least two species could be asThe reaction between aerobic Y122F apoprotein R2 and ansigned to tryptophan free radicals. Thisis the first EPR aerobic ferrous ammonium sulfate was allowed to proceed at observation of relatively stable protein-linked trypto- 25 "C for a specified time, whereupon EPR spectra were obphan radicals at room temperature and at 77 K. These served either at 77 K or a t room temperature. Fig. la shows the tryptophan radicals may be involvedas redox interme- EPR spectrum observed at 77 K after 6 s of reaction time. It is diates in long range electron transfer within the protein a composite spectrum withtwo major components arising from structure. two different species as indicated in the figure legend, separable by different microwave saturation behavior and decay kinetics (data notshown). The total concentration of unpaired The small subunit of iron-containing ribonucleotide reducspins in the spectrum, quantitatedby comparing with a Cu2+/ tases, protein R2, contains in its active state a p-oxo-bridged EDTA standard, is about 20% of the proteinconcentration. One diferric iron center and a stable free radical on a tyrosyl residue spectral component (I) has the characterof an axial spectrum (Tyr'") in Escherichia coli (1-5). The in vitro formation of the with g = 2.036 and g = 2.009. The othermajor component (11)is irodradicalsite from apoprotein R2 is a complex reaction a 1:l:l:l hyperfine quartet with coupling constants 28 and13 which may be summarized as, G, a t g = 2.004. Fig. l b shows the corresponding EPRspectrumobtained %(P) + 2FeZ++ e + 0, + H' (Tyr(P)/Fe2+ . . . Fez+)+ from a Y122F R2 protein containing indole-d,-Trp. There is a Tyr'(P)/Fe3+-02--Fe3+ + H,O clearly noticeableisotope effect on spectral component 11. It can where Tyr(P) denotes the apoprotein, m P ) / F e 2 +. . . Fez+ the be explained by a decreased line width in the spectrum, due to fully reduced protein, andTyr'(P)/Fe3+-OZ--Fe3+ the active state the disappearance of minor unresolved hyperfine couplings. This shows that component I1 is associated with a radical on a (6-8). The tyrosyl radical, about 5 A from the nearest ironion, "p residue, with spin density in the indole ring. Since the ring protons are substitutedby deuterium, themajor hyperfine cou* T h i s study was supported by grants from the Swedish Natural ScienceResearchCouncil, the Carl Trygger Foundation, the Magn. plingfs) must arise from the p-methyleneprotons and/or possiBergwall Foundation, and the Deutsche Forschungsgemeinschaft,Pro- bly the ringnitrogen. The involvement of nitrogen is, however, ject 751/1-1. The costs of publication of this article were defrayed in less likely, judging from the spectral shapes. The shape of compart by the payment of page charges. This article must therefore be ponent I is not significantly affected by incorporation of the hereby marked "advertisement"in accordance with 18 U.S.C. Section deuterated Trp. Both spectral components I and I1 decay on the 1734 solelyto indicate this fact. ** To whom correspondence should be addressed. Tel.: 46-8-162450; Fax: 46-8-155597. M. Karlsson, personal communication.

Tryptophan Radicals Formed by IrodOxygen Reaction with Escherichia coli Ribonucleotide Reductase Protein R2 Mutant Y122F*

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%ptophan Radicals in Mutant Protein R2

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3520 [G:

FIG.2. First derivative EPR spectrum at room temperature. FIG.1. First derivative EPR spectrum at 77 K of the intermediates after 6 s of incubation of 100 q aerobic apo-Y122F R2 Stopped-flow EPR (15)was used tomix equal volumesof aerobic 100 PM Fez+.a shows the spectrum after with 400 q anaerobic Fez* ammonium sulfate. Equal volumes (75 apo-Y122F R2 with anaerobic 400 111) of apo-R2 and Fez+ weremixed i n a nEPR tube with a 3-mm inner the reaction of apo-Y122F grown in normal medium.b shows the specdiameter, and the reaction was quenched by freezing with cold isopen- trum after thereaction of apo-Y122F from cells grownin medium with of 16 scans, and the tane (170 K). a shows the spectrum after the reaction of apo-Y122F indole-d, tryptophan. Both spectra are the sum grown in normal medium. b shows the spectrum after the reaction of corresponding backgroundhas been subtracted. Spectra wererecorded 1-12 min after shot.Arrows indicate two hyperfine apo-Y122F from cells grown in medium withindole-d, tryptophan. Ar- in the time interval rows i n a indicate theg, and g , for component I. Arrows in b indicate the components separatedby 19 G i n a and 17 G in b. Recording conditions: modulation amplitude, 3 G microwave power, 40 milliwatts; microwave hyperfine lines and g value of component 11. The two components were frequency, 9.7874 GHz. separated by partial subtractions of spectra, based on their different behavior with regard t o microwave saturation and time-dependentdecay. Recording conditions:modulationamplitude, 3 G; microwave power, 4 milliwatts; microwave frequency, 9.4902 GHz. No background incorporation of indole-d,-Trp may be tentatively assigned to has been subtracted. oxidation radicals of Trp. Plausible Trp candidFtes close to the

minute timescale at room temperature; after 10 min of reaction time, component I is the dominant species, which in turn is essentially decayed after another 10 min. Microwave saturation studies on these spectral components show that they are not easily saturated a t 77 K (data not shown), which in turn suggests that they havea fairly close interaction with a metal site. Hence, we expect their EPR spectrato be severely broadened and difficult to observe at room temperature. Fig. 2a shows that a prominent EPR spectrum of Y122F protein R2 is obtained a t room temperature aftera few minutes of reaction time. It is characterized by a doublet hyperfine splitting of 19 G. The g value is 2.004. Fig. 26 shows the corresponding spectrum obtained in a Y122F protein containing indole-d,-Trp. Again there is a clear isotope effect. The line width in the deuterium-containing sample is decreased. This spectral component, which can be found as a minor contributor also to thelow temperature spectra after suitably long reaction times, must arise from a Trp-based radical with small or negligible metal interaction. This is consistent with a rather long distance between ironcenter and tryptophan radical. Again the hyperfine coupling pattern should involve the p-methylene protons. Theroom temperature doublet spectrum starts to appear after about 10 s of reaction time and has its maximum after about 3 min. This radical is relatively long lived (20 min or more). The radicals assigned to Trp and observed at 77 K or at room temperature must belong to different species. The reason for this conclusion is mainly kinetic. The quartet spectrum observed a t 77 K appears with full intensity already after 6 s of reaction time, whereas thedoublet spectrum observed at room temperatureappearsafter longer reaction times,starting around 10 s and growing during a minute or more (data not shown). Thequartet essentially disappearedafter 10min, whereas thedoublet remained observable after up to30 min of reaction time. Both paramagnetic species showing isotope effects due to

iron center in the protein are: (i) Trp4', -8 A from Fel and participating in a H-bonding network with theiron ligand histidine 118 (Fel-His"s-Asp237-Trp48), (ii) Trp"', about 4 A from Fe2 and at H-bonding distance from the iron ligand glutamic acid 204 (Fe2-Gl~~~~-Trp"'), andTrp107, (iii) -8 A from Fe2 and H-bonded to a water molecule that in turn isH-bonded to the carbonyl group of the iron ligandhistidine 241 (F~Z-H~S~*~-H,OTrp1O7).In addition the indole rings of Trp107and Trp"' are in van der Waals contact with each other. The positioning of the three tryptophans inrelation to the iron center of R2 is shown in Fig. 3. The iron-linked amino acid triad (His-A~p-Trp~~) is absolutely conserved among all protein R2 species described so far and hasbeen suggested to take part inlong range electron transport between the substrate binding site on the large subunit and the irodradical siteprotein in R2 during the catalytic reaction (9, 10). The other two tryptophans areonly present in E. coli and bacteriophage T4 R2. At the position of Trplo7is either tyrosine or phenylalanine in the other species, and at the position of Trp"' is in all other species glutamine, which has similar H-bonding capacity as tryptophan. Previous studies of the reaction of wild type apoprotein R2 with Fez+ and0, have shown other intermediates on the subsecond time scale, characterized by various kinetic spectroscopic techniques (8, 16). The first intermediate, characterized by Mossbauer spectroscopy and lightabsorption, was suggested to be a p-peroxodiferric complex (8) or a diferric radical together with a Trp cation radical, the latter responsible for the light absorption (16). A later intermediate, characterized by light absorption and EPR,was suggested t o be an iron-coupled free radical ( X ) .These intermediateswere alsoobserved in the Y122F mutant, where they were found to be more long lived than in thewild type protein. Also in the mutantprotein they were, however, all gone after a few seconds of reaction time at 5 "C. At shorter reaction times than dealt with in the present study (