Kinetic studies on the reduction of the tyrosyl radical of the R2 subunit of E. coli ribonucleotide reductase

BB

Biochimicaet BiophysicaActa 1247 (1995) 215-224

ELSEVIER

etBiochi~ic~a Biophysica/~ta

Kinetic studies on the reduction of the tyrosyl radical of the R2 subunit of E. coli ribonucleotide reductase Jannie C. Swans, Manuel A.S. Aquino, Joo-Yeon Han, Kin-Yu Lam, A. Geoffrey Sykes

*

Department of Chemistry, The University of Newcastle, Newcastle upon Tyne, NE1 7RU, UK

Received 28 February 1994; revised 21 October 1994; accepted29 November1994

Abstract

Kinetic studies at 25°C, I = 0.100 M (NaCI), on the reduction of the tyrosyl radical of the R2 protein of E. coli ribonucleotide reductase with hydroxyurea (HU), N-methylhydroxylamine, catechol, and seven hydroxamic acid derivatives are reported. There are no pH-dependences in the range 6.2-8.6 investigated except that introduced with N-methylhydroxylamine which itself protonates in this range. At pH 7.6 the rate constant (0.46 M - t s-1) for the HU reaction is in agreement with earlier values. Slower reactions are observed with the bulkier acetohydroxamic (0.020 M - t s- 1) and benzohydroxamic acids (0.040 M - 1 s- t). In the case of N-methylhydroxylamine the rate constant (0.41 M-1 s-1 at pH 7.6) decreases with pH, and it is concluded that the protonated form CH3NH~-OH ( p K a = 6.2) has little or no reactivity with Tyr ". For this reaction under air-frce conditions a second-stage (0.027 M-1 s-1) corresponding to reduction of Fe(III)2 is observed. Mid-point redox potentials for the reductants and estimates of reduction potentials applying in the case of the protein are considered. The reactions with 1,2-dihydroxybenzene (catechol) and 3,4-dihydroxybenzohydroxamic acid (Didox) also have two stages, when the initial Tyr" reduction, rate constants/M-1 s-1 for catechol (3.2) and Didox (0.010), is followed by removal of the Fe(III) to give catechol and catechol like Fe(III)-complexedproducts. The single stage reactions of the hydroxamic acid derivatives which incorporate charged amino-acid groups L-glutamic acid, L-histidine, L-glycine and L-lysine, are slow, and saturation kinetics are observed consistent with association (small K values) prior to redox. The mechanism of reduction of R2-Tyr" by all of the reagents studied is discussed. 1. Introduction

Ribonucleotide reductase (RNR) catalyses the first unique step in the synthesis of DNA monomer forms by reducing ribonucleoside diphosphates to deoxyribonucleotides (1), [1-3].

RNR

OH H r'i bonucleotide

deoxyr'ibonucleotide

(1) The enzyme isolated from E. coli is the best characterised RNR to date, and is regarded as a prototype of the enzyme found in eukaryotes [4-6], viruses [7], many prokaryotic organisms [8,9], and bacteriophages [10,11].

* Corresponding author. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All fights reserved SSDI 0167-4838(94)0023";-9

The enzyme from E. coli consists of homodimeric subunits R1 (formerly B1) and R2 (formerly B2), molecular weights 2 × 85.5 kDa and 2 × 43.5 kDa respectively. Each R2 monomer (375 amino acids) has a binuclear /z-oxo iron(Ill) centre, the function of which is to stabilise a tyrosyl radical at residue 122 required for enzymic activity. The structure of the R2 homodimer, which is a heart shape has been determined to 2.2 A, resolution [12], and that of R1 has recently been reported to 2.7 A [13]. The R1 protein binds all four ribonucleoside diphosphate substrates. A feature of R1 is the involvement of cysteine residues in the redox steps leading to deoxyribonucleotide formation, [14-18]. The R1 and R2 proteins associate in the presence of Mg 2+. A number of papers have appeared in which the combined involvement of R1 and R2 has been considered, [19-23]. The crystal structure of E. coli R2 in the inactive met form has demonstrated that Tyr-122 is embedded in the protein, and is ~ 10 ~, away from the upper surface of the heart shape which is believed to be the site of interaction with R1 [12]. The Tyr" radical is in a phenolate form with the phenolic H-atom removed [24,25]. The phenolate group

216

J.C. Swarts et aL /Biochimica et Biophysica Acta 1247 (1995) 215-224

Y122~

O "

D84N~ ~/ "

O

H200 " ~ E23B / E)

.............. Io.-7ie"'o

l

Hl18.,.,c~N\

t,

T /N',,~f H241

Fig. 1. The active site of active E. coli R2 ribonucleotide reductase from

the X-raycrystalstructureof the metR2 form (Ref. [12]). is 5.3 .~ away from the nearest Fe atom, and located along the Fe-Fe direction, Fig. 1. The Fe atoms are at about the same distance from the surface. Although R2 has the potential to form two Tyr" radicals per dimer, reports to date indicate a maximum 1.5, [26], and 100% active R2 has not yet been detected. Stable radical formation is not observed in the absence of the Fe(III) 2 centre, which is required for enzymatic activity. However, the inactive metR2 form is obtained by treating R2 with hydroxyurea or other radical scavengers, and the Fe(III) 2 centre can exist without the radical. Since the Tyr" of R2 is relevant in the reduction of ribonucleotides, which in turn is an essential step in de novo synthesis of DNA monomer forms, it has become a target in anti-cancer, anti-bacterial and anti-viral drug action [2,27]. Here in the first of a series of papers concerned with the kinetic reactivity of the buried Tyr" and Fe(III) 2 centres we report studies on the reduction of the T y r of R2 with hydroxyurea, N-methylhydroxylamine, catechol and a number of hydroxamic acid derivatives. Earlier reports including a preliminary account of this work have appeared [28-34], but so far no systematic kinetic studies on RNR reactivity have been published. In the reaction with N-methylhydroxylamine under air-free conditions two stages are observed corresponding to reduction of Tyr--* TyrH (tyrosine), and Fe(III) 2 ---)Fe(II) 2. For reactions carried out in the presence of 0 2 spontaneous regeneration of T y r and Fe(III) 2 occurs, and only the more rapid T y r reduction is observed until the 0 2 present is used up.

Department of Molecular Biology, University of Stockholm, S-10691 Stockholm, Sweden. This was used to grow E. coli cells overproducing R2. The growing and isolation procedures for R2 were as described previously [35,36]. Growing was carried out in the University Department of Microbiology, where all safety regulations and requirements of the University Genetic Manipulation Committee were conformed to. In the procedure used reactivation to increase the T y r content of R2 was carried out according to [37] before desalting. Concentrated protein solutions from a DE52 column were purified finally on an FPLC HR 5 / 5 Mono Q anion-exchange column (1 ml bed volume) at room temperature. The gradient was developed with solutions of air-free ice-cooled 50 mM Tris pH 7.6 (A) and 50 mM Tris, pH 7.6 in 1.0 M NaC1 (BDH, Analar) (B). A typical elution profile is shown in Fig. 2. The fractions collected at about 28% B were ultradialysed air-free against 50 mM Tris (pH 7.6) with 20% glycerol. Another peak was observed at = 26% B. This became more pronounced upon ageing of the protein, and also when cooling was not applied during the FPLC. It shows no obvious differences in its UV spectrum when compared with the previous peak, but it gives rate constants, e.g., with acetohydroxamic and benzohydroxamic acids, which are substantially different. Typically from 50-60 g of E. coli cells the yield of R2 was 250 mg. The concentrated (green) protein solution ( = 0.3 mM) containing ca. 200 mg of R2, could be stored for long periods at -80°C, but more dilute solutions (50/xM) for only < 2 weeks. The activity of R2 was in satisfactory agreement with literature values [35,36], and the Tyr' content was determined from the UV-Vis absorbance at 410 nm. To prepare MetR2, a concentrated solution (1-2 cm 3) of active R2 ( = 0.50 mM) was incubated with hydroxyurea ( = 100 mM) at 25°C for 30 min. The solution was then dialysed against two portions of 50 mM Tris buffer containing 20% glycerol at pH 7.5. The volume of each portion was at least 200 × that of the protein solution. 1

100

o;

=

2. Materials and methods 2.1. R 2 Protein

The R2 subunit of ribonucleotide reductase used in this work was isolated from a genetically modified strain of E. coli as described [35,36]. The resultant overproducer, PBS1, which is named according to the recombinant plasmid construct, was provided by Professor B.-M. Sjfberg,

0

o

1o

20 30 eluant volume / cm s

40

Fig. 2. FPLC Mono Q purification of active E. coli P,2 ribonucleotide reductase using buffers A (50 m M Tris-HC1 at pH 7.6), and B (50 m M Tris-HCl at pH 7.6 in 1.0 M NaCI) as gradient. The flow rate was 2 cm3/min, /'max < 3 MPa. The absorbance peak at 280 nm labelled a

correspondsto pure enzymeand b to an impure fraction.

217

J.C. Swarts et al./Biochimica et Biophysica Acta 1247 (1995) 215-224 2.2. Reagents

Hydroxyurea, acetohydroxamic acid, benzohydroxamic acid, N-methylhydroxylamine, L-glutamic acid-3,-monohydroxamate, glycine-hydroxamate, L-histidine hydroxamate and L-lysine hydroxamate hydrochloride were obtained from the Sigma, and 1,2-dihydroxybenzene (catechol) from Aldrich. The reagent 3,4.-dihydroxybenzohydroxamic acid (Didox) was generously provided by Dr. W.R. Vezin from the Cancer Research Campaign Laboratories in the Department of Pharmacy at the University of Strathclyde, Glasgow.

12

L

2.3. p H and buffers

The pH of reactant solutions containing as buffer either tris(hydroxymethyl)aminomethane ('Iris, pH range 7.09.1), or 2-(N-morpholino)ethanesulfonic acid (Mes, pH 5.2-6.9) at 50 mM was confirmed after completion of kinetic runs on a Radiometer-PHM62 pH-meter fitted with a Russell CWR-322 glass; electrode.

0 300

I 400 Wovelength

2.4. Kinetic studies

All studies were carried out at 25.0 + 0.1°C with the pH adjusted to values in the range 6.2-8.6. The ionic strength I was adjusted to 0.100 + 0.001 M with NaCI (BDH, Analar). Solutions for anaerobic studies were prepared in a Miller-Howe glove-box, 0 2 level < 5 ppm. The procedure for kinetic runs involved thermostatting 1.1 cm 3 of reductant in a narrow 1 cm path length spectrophotometer cell. The R2 solution (0.05 cm 3) was then added using a Gilson Pipetman, and mixing carried out with non-vigorous shaking. Final R2 concentrations were ~ 10 /zM. Reactions involving the reduction of Tyr" were monitored at 410 nm (~ 6600 M -1 cm-m), [1,37], while combined Tyr" and Fe(III) z reduction was generally monitored at 370 nm (~ 8900 M -1 cm -1) a n d / o r 410 nm, Fig. 3. Chelation of Fe(III) by catechol and Didox were monitored at wavelengths of 500 and 492 nm respectively. Reactions of N-methylhydroxylamine, were studied anaerobically in order to monitor two stages, as were also the catechol and Didox reactions, since (at the higher pH values) 0 2 oxidises these reagents to quinones which contribute to the absorbance changes. The reactions of other hydroxamic acid derivatives and HU were found to be unaffected by the presence of air. For each reagent, scan spectra (300-500 nm) were recorded at appropriate time intervals in order to identify whether Tyr" a n d / o r Fe(III) z are involved in the reaction. The slopes of first-order plots of ln(A t - A = ) against time were linear for at least two half-lives, and gave rate constants klobv In the case of the N-methylhydroxylamine, catechol and Didox reactions biphasic behaviour was observed. The above plot gives kzob~ (latter stages). To obtain k~ob~ for the earlier stages the intercept x at t = 0

I 500 / nrn

Fig. 3. UV-Vis spectra of different oxidation states of E. coil R2 ribonucleotide reductase: the active (fully oxidised) Tyr and Fe(III)2 containingprotein(--), the metR2formin which Tyr but not Fe(HI)2 is reduced (---), and the fully reducedtyrosine(TyrH)and Fe(II)2 containing protein (. - - ).

and slope kzobs allow a modified plot of in[A t - A ® xexp(--kzobst)] vs. time to be carried out. Second-order rate constants were obtained from the dependencies of klob~ and k2obs against reductant concentration. Conventional time range studies were monitored on either Shimadzu UV-2101PC or Perkin-Elmer Lambda 9 spectrophotometers. A Dionex D-110 stopped-flow spectrophotometer was employed to monitor the reduction of R2 with HU at the higher concentrations. 2.5. Reaction products and stoichiometries

The optimum concentration range for R2 used in these experiments was 10-20 ~M. The reductants hydroxyurea, N-methylhydroxylamine and hydroxamic acid derivatives (RNHOH) are expected to yield directly or indirectly radical-like products in reaction steps as in (2), RNHOH + R2-Tyr'-* R N H O ' + R 2 - T y r n

(2)

where R2-Tyr" denotes the tyrosyl radical of R2. From EPR stopped-flow studies on the hydroxyurea reaction Lassmann et al. [32] have demonstrated that nitroxide-like radicals NH2CONHO" are produced. Subsequent rapid dimerisation a n d / o r disproportionation of these radical to stable products occurs (stoichiometry 1:1). Visible range absorbance spectra of the products obtained in the reactions with catechol and Didox correspond to those of known Fe(III) products.

J.C. Swarts et al. /Biochimica et Biophysica Acta 1247 (1995) 215-224

218

Table 1 The dependence of first-order rate constants klobs (25°C) for the reduction of the tyrosyl radical Tyr' of E. coli R2 ribonucleotide reductase on reductant concentration at pH 7.6 (50 mM Tris), I = 0.100 M (NaC1) Reductant

Hydroxyurea, NH2CONHOH [HU]/mM 5.0 10.0 18.8 103klobs/S - 1 2.7 3.8 8.6 Acetohydroxamic acid, CH3CONHOH [AcetoHA]/mM 15.0 28.5 42 104klobs/S - 1 3.1 6.0 9.0 Benzohydroxamic acid, C6 Hs CONHOH [BenzoHA]/mM 20 30 40 104klobs/S - 1 8.4 12.3 17.2 N-Methylhydroxylamine, CH3NHOH [CH3NHOH]/mM 0.65 6.5 11.0 103klobs(lO3k2obs/S - 1) 0.32 ( - - ) 3.2 (0.18) 4.7 (0.34) 1,2-Dihydroxybenzene (Catechol), (OH)2C6 H 4 [Catechol]/mM 1.4 2.4 5.2 103klobs(lOSk2obs)/S - 1 4.0 (1.7) 7.5 ( -- ) 16.7 (2.8) [Catechol]/mM 30.5 61.6 lOaklobs(lO5k2obs)/s - 1 - (15.5) - (63.3) 3,4-Dihydroxybenzohydroxamic acid (Didox), (OH)2C 6 H3CONHOH [Didox]/mM 13 43 86 105klobs(lOSk2obs)/S - 1 12 (4.0) 43 (5.6) 89 (29) L-Glutamic acid- ?l-monohydroxaraate, - OzCCH(NH~ )(CH2 )2CONHOH [GluHA]/mM 10.3 18.7 23.3 103klobs/S- 1 0.7 1.14 1.48

25.0 11.7

30 13.6

51 10.0

55 11.0

48 19.0

50 20.7

16.7 7.0 (0.46)

20.0 a 8.1 ( - )

50 21

71 33

85 38

100 45

150 70

10.4 34 (3.0)

95 103 (38) 65 3.5

96 4.3

15.7 6.2

a In air, no k2obs.

2.6. E l e c t r o c h e m i s t r y

working electrode against an Ag/AgC1 ence

A

three-electrode system

was

used,

electrode.

voltammograms

consisting of a

Attempts and

were

(1 M K C I ) r e f e r -

made

square-wave

to

obtain

cyclic

voltammograms

at

-~ 2 5 ° C f o r h y d r o x y u r e a , a c e t o h y d r o x a m i c a c i d , b e n z o b y -

Pt-wire auxiliary electrode and an EG and G glassy carbon

Table 2 Second-order rate constants k 1 (25°C) for reduction of the tyrosyl radical Tyr of E. coli R2 ribonucleotide reductase, at different pH values (50 mM buffer: Mes at pH < 6.5 and Tris at pH > 6.5), I = 0.100 M (NaC1) Reductant

Hydroxyurea, NH2CONHOH pH 6.40 k l / M -1 s - 1 0.46 Acetohydroxamic acid, CH~CONHOH pH 7.10 102kl/M - 1 s - 1 1.9 Benzohydroxamic acid, C6H 5CONHOH pH 6.51 102kl/M - 1 s - 1 4.0 N-Methylhydroxylamine, CH3NHOH pH 6.21 10kl(10k 2 ) / M - 1 S-1 2.1 (0.27) pH 6.71 lOkl(lOkE)/M -1 s-1 - (0.25) pH 7.10 lOkl(lOk2)/M -1 s - ~ 3.8 (0.28) 1,2-Dihydroxybenzene (Catechol), (OH)2C6H ~ pH 6.40 kl(lO2k2)/M - 1 s - 1 2.7 ( - ) 3,4-Dihydroxybenzohydroxamic acid (Didox), (OH)2C6H3CONHOH pH 6.40 103kl(103k2)/M - x s - 1 9.7 (1.3) L-Glutamic acid- ~,-monohydroxamate, - 0 2CCH(NH~ )(CH2 )2CONHOH pH 6.40 102kl/M - 1 s - 1 5.0

6.67 0.46

7.10 0.46

8.45 0.47

7.60 2.0

8.20 2.0

8.60 2.0

7.10 4.0

7.60 4.0

8.60 4.1

6.40 2.4 (0.29) 6.90 3.4 (0.27) 7.60 4.2 (0.28)

6.51 2.6 (0.27)

8.40 4.2 (0.30)

7.60 3.2 (0.76)

8.40 3.1 (2.0)

7.60 9.7 (1.3)

8.40 6.5 (1.2)

7.10 6.0

8.45 6.0

219

J.C. Swarts et al. / Biochiraica et Biophysica Acta 1247 (1995) 215-224 2.7. Treatment o f data

0.4

0.2

The program MINSQ was used to evaluate rate constants when there was some uncertainty as to final absorbance (A®) values. To obtain second-order rate constants linear least-square fitting procedures weighted to pass through the origin and in one case (the saturation kinetic studies with glutamic acid-T-hydroxamate) an unweighted non-linear least-squares procedure was used.

S

/

3. Results 3.1. Hydroxyurea as reductant • O 6.0

*

= I

= I 7.5

i

{"

I 9.0

pH

Fig. 4. The dependence of sccc,nd-order rate constants (25°C) on pH for the reduction of the tyrosyl radical of active E. coil R2 ribonucleotide reductase subunit with different reagents, I = 0.100 M (NaCl), hydroxyurea (,), acetohydroxamic acid (O), benzohydroxamic acid (11), Nmcthylhydroxylamine ( • ) , 3,4-.dihydroxybcazohydroxamic acid (Didox) ( # ) , L-glutamic acid-'y-monohydroxamate ( • ).

droxamic acid, N-methylthydroxylamine, L-glutamic acidT-hydroxamate, catechol and Didox, in 50 m M Tris buffer solutions at pH 7.5, I = 0.10 M (NaCI). In none of these was reversible behaviour observed except catechol. Midpoint redox potentials (Esw v in mV vs. normal hydrogen electrode) from square-wave voltammograms (SWV) were recorded as a measure of their comparative reductive capability. The values obtained are for hydroxyurea (1070), acetohydroxamic acid (1035), benzohydroxamic acid (58), N-methylhydroxylamine ( = 1130), L-glutamic acid-T-hydroxamate (1155) and Didox (435). In the case of catechol the E ~ value is 373 mV from both cyclic voltammetry and square-wave voltammet D' measurements.

First-order rate constants klobs (25°C) at pH 7.6 with hydroxyurea (HU) in > 10-fold excess are listed in Table 1. Concentrations of hydroxyurea > 50 m M required the use of the stopped-flow method. Results confirmed the applicability of the rate law (3) over the extended range of [HU], with no evidence for saturation kinetics. Rate = klobs[RE-Tyr'] = kl [HU] [RE-Tyr ']

(3)

From a least-squares treatment the second-order rate constant k 1 is 0.46 + 0.01 M -1 s -1. There were no contributions from the spontaneous decay of R2-Tyr" at 410 nm over the same reaction times. No effect of pH on k~ was detected over the range 6.4-8.5, Table 2. The initial reaction, (4), NHECONHOH q- R2-Tyr" NHECONHO'+ R2-Tyrn

(4)

is followed by further rapid step(s) as indicated in the experimental section. 3.2. Aceto- and benzohydroxamic acids as redactants

Rate constants klobs for aceto- and benzohydroxamic acid are listed in Table 1. The highest pH used (8.6) is less

Table 3 Summary of second-order rate constants k1 (25°C) for the reduction of the tyrosyl radical Tyr of E. coil R2 dbonucleotide reductase, with different reagents at pH 7.6, I = 0.100 M (NaCI) Reductant Formula k l / M -1 s- 1 Hydroxyurea Acetohydroxamicacid Benzohydroxamicacid N-Methylhydroxylamine 1,2-Dihydroxybenzene a 3,4-Dihydroxybenzohydroxamicacid b L-Glutamic-T-monohydroxamatec L-Histidine hydroxamate e L-Lysinehydroxamate c Glycine hydroxamate c

NHECONHOH CH 3CONHOH C6HsCONHOH CH3NHOH (OH)EC6H4 (OH)EC6H3CONHOH

0.46 0.020 0.040 0.41 3.2 0.010

- OECCH(NH ~ X C H E ) E C O N H O H ( C a N EH a ) C H E C H ( N H ~ ) C O N H O H

0.068 d 0.0044 e

+NH 3(CHE)4CH(NH+)CONHOH + NH3CHECONHOH

0.0041 e 0.0083 e

a Catechol. b Didox. c Names as used in Sigma cata~[ogue.Alternatives, e.g. L-glutamate-T-monohydroxamicacid, are sometimes used. d From the slope of the plot in Fig. 8 at the lower reductant concentrations. e Using klobs for lowest reductant concentration.

220

J.C. Swarts et aL / Biochimica et Biophysica Acta 1247 (1995) 215-224

than values at which acid the dissociation (5), RCONHOH ~ R C O N H O - + H +

0.2

(5)

is effective [38,39]. At pH 9.0 R2 (10-16 /xM) denatures giving a white flocculence within 2 min, and at pH 8.7 solutions are stable for only = 1 h. At pH values up to 8.6 no effects of pH on rate constants were detected, Table 2. Values of k I obtained are 0.020 _ 0.001 M -1 s-1 (aceto-), and 0.040 + 0.002 M - 1 s - 1 (benzo-). ¢J u c

3.3. N-Methylhydroxylamine as reductant

8 At pH 7.6 under anaerobic conditions two stages klobs and k2obs are observed, Table 1, both of which exhibit linear dependencies on [CHaNHOH]. The second-order rate constant k 1, Table 2, is pH dependent, Fig. 4, and protonation/deprotonation (6)

0.1

.Q

,<

Ka

CH3NH~OH ~

CH3NHOH + H +

(6)

is implicated. Two rate constants can be defined (7) and (8), ko

CH3NHOH + R2-Tyr' ~

I 400

0

C H 3 N H O ' + R2-TyrH

(7)

I 500

I 600

I 700

Wavelength / nrn

kr[

C H a N H ~ O H + R 2 - T y r --* C H 3 N H ~ O + R2-TyrH (8) from which the expression for kl, (9), is obtained. k1=

koKa + kH[H +] ( K a + [H+] )

(9)

From a non-linear least-squares fit of k I at different [H ÷] values p K a = 6.4 ___0.2, ko = 0.41 + 0.01 M -1 s -1, and k H = 0.05 _ 0.08 M -1 s -1. The latter is zero within experimental error. The k o value, i.e., k 1 at the higher pH values, is listed in Table 3. We regard the pK~ of 6.2 __+0.1 determined by potentiometric titration against NaOH as more accurate than that from kinetics because the latter studies were restricted to pH > 6.7. From the spectrophotometric changes, Fig. 3, the second stage of reaction k2obs corresponds to reduction of Fe(III)2. The rate constants (Table 1) give a first-order dependence on [CH3NHOH], and k 2 = (2.7 + 0.2)- 10 -2 M -1 s -1. The dependence of k2obs on pH is less clearcut. For a run with air present the first stage is complete in = 50 min, and no k2ob~ stage is observed up to 700 min of reaction time until the 02 is used up in the regeneration of active R2. A further decrease in absorbance is then observed.

Fig. 5. Visible range absorbance scan spectra for the two stage reaction (25°C) of Didox, (OH)2C6H3CONHOH (86 raM) with E. coli R2 ribonucleotide reductase (10 gM) at pH 7.6, I = 0.100 M (NaC1). The seven lower traces are separated by 5 min, and the upper traces by 30 rain intervals. Loss of the tyrosyl absorbance at 410 nm (first stage), is followed by Didox scavenging complexing of the Fe(III) (absorbance increase peak at 492 nm).

monitored at 410 nm corresponds to reduction of Tyr" and is soon overlaid by the second stage. The latter change gives much more intense absorbance bands with peaks centering at 500 nm in the case of catechol and 492 nm for Didox characteristic of the formation of Fe(III)-catecholate complexes. Stepwise formation constants at pH 7.4 for the complexing of catechol with iron(Ill) to give complex A, (10 20"0, 1014"7 and 109"1), are more favourable than those for acetohydroxamic acid to give complex B, (1011"4, 109.7 and 107"2) [40], consistent with a predominance of A rather than B.

0

v

-0-_i 3

3.4. Reactions of R2 with catechol and didox Both reagents have ortho-dihydroxy functional groups which can be redox active and/or complex to metal ions. Didox also has a hydroxamic acid group which has similar properties. Scan spectra for both reagents indicate two stages of reaction, e.g., Fig. 5 for Didox. The first stage

3-

(A)

H~

N ~0

~J3

(B)

First:order rate constants klobs for the T y r reductions are listed in Table 1. These give dependences on [catechol] and [Didox] respectively, rate law as in (3). A substantially

J.C. Swarts et al. / Biochiraica et Biophysica Acta 1247 (1995) 215-224

221

faster reaction ( × 300) is observed in the case of catechol, k I = 3.2 + 0.1 M -1 s -1, than Didox k 1 -- 0.0104 _+ 0.0002 M -1 s -1. No dependence of klobs values on pH is observed, Table 2. Acid dissociation constants p K , have been determined for catechol and are 12.8 and 9.4 at I = 0.10 M (KCI) [41]. Iqae second stages k2obs were also studied, but evaluation of rate constants is complicated by the presence of mono, bis- and tris-chelated Fe(III) complexes (with different associated E values). This results in apparent dependences on the reductant of power between 2 and 3, which have no meaning and are regarded as spurious. Rate constants obtained for the second stage of the reaction with Didox monitored at different wavelengths 370, 410 and 492 nm were found to be identical. At 43 mM reagent concentrations (pH 7.6), rate constants k2obs are 2 . 8 . 1 0 -4 s -1 (catec!hol) and 0 . 5 6 . 1 0 -4 s -1 (Didox), suggesting a 5 × more favourable catechol reaction. Runs carried out on the reaction of met-R2 with Didox, gave rate constants 105k2obJS - 1 ([Didox]/mM in brackets) of 5.3 (43), 11.0 (66), 17.3 (81) which are upto 30% smaller than those listed in Table 1.

Fig. 6 gives a second-order rate constant corresponding to k I ( = k r K ) of 0.068 M -1 s -1.

3.5. Reaction with L-glutamic acid-7-monohydroxamate

The reactions of ten reductants with the E. coli R2 subunit of ribonucleotide reductase are reported in this paper. Of these seven give reduction of the Tyr" only, one gives reduction of Tyr" and Fe(III) 2 when studied under anaerobic conditions, and two give reduction of the Tyr" followed by removal (by chelation) of the Fe(III) from R2. Thus three of the five categories of reaction previously defined [42] are represented. A prime interest is in the reduction of the T y r which is common to all the reactions. Most earlier rate studies on the reduction of ribonucleotide reductase, have recorded only tx/2 values, which may of course lead to important features being overlooked. An exception is the reaction with hydroxyurea which has been studied by a number of groups and serves as a marker. The rate constant 0.46 M-1 s-1 obtained at pH 7.6 is in good agreement with previous values in the range 0.43-0.50 M -1 s -1 [31,32]. There are no pH dependences in the range 6.2-8.6 investigated except that introduced by N-methylhydroxylamine, which gives a protonation constant p K a 6.2. No pH dependences are detected originating from the R2 protein. Therefore, the pH dependence in the case of the methylhydroxylamine is explained by the protonated form CH3NH~OH having little or no reactivity with the Tyr" of R2. This suggests that charge inhibits association of the reductant at the protein surface or penetration to access the active centre whichever applies [28]. It is also in line with the inability of dithionite, with reduction potentials for the SO~-/HSO~- and S 2 0 2 - / H S O 3 couples reported as - 660 mV and - 180 mV respectively (there are other values) at pH 7.0, [43,44], to reduce the Tyr" of R2 in the absence of a mediator, [45]. The reaction with [Co(sep)] 2+ ( - 3 0 0 mV), is also very slow [46], suggesting again that a charged reactant, however good a reductant, has difficulty in reducing the active centre corn-

A single stage pH-independent reaction is observed. Values of klobs, Table 1, give a non-linear dependence on [GIuHA], Fig. 6. The latter is consistent with a mechanism involving saturation kinetics, (10)-(11). r GluHA + R2-Tyr" ~ R2-Tyr',GIuHA

(10)

kr

R2-Tyr ',GluHA ~ R2-TyrH + GluA"

(11)

The rate dependence (12) can be derived klobs = k r K [ G l u H A ] / ( 1 + K [ G l u H A ] )

(12)

which yields K = 5.6 + 0.7 M-1, and rate constant for the redox step of 102k, = 1.3 + 0.2 s -1. The initial slope in

0.6

~"

14

~0.3

[GluHA]-' / M ~

o

'

'

' o.~a [GluHA] / M '

'

' oJ6

Fig. 6. The dependenceof rate constants klobs (25°C) for the L-Glutamic acid-7-monohydroxamate, - OzCCH(NH~ XCH2)2CONHOH (GluHA), reduction of the tyrosylradical of active E. coli ribonucleotidereductase (410 nm) at pH 7.6, I = 0.100 M (NaCI). A linear reciprocal plot is obtained (inse0 consistent with saturation kinetic behaviour.

3.6. Other reactions The reactions of three other amino-acid hydroxamate derivatives containing respectively L-histidine, L-lysine and glycine, were studied briefly. The reactions are all slow and give rate constants 105kobs/S -1 (concentrations of reductant/mM in brackets): L-histidine 5.2 (11.9), 7.0 (26.9); L-lysine 11 (26.6), 15 (51); glycine 25 (30), 36 (109). The low solubility of the L-histidine derivative restricted the range of concentrations that could be used. From this limited information it would appear that like the L-glutamic acid derivative all three reagents exhibit behaviour consistent with saturation kinetics. The data reported do not allow accurate K and k r values to be determined.

4. Discussion

222

J.C. Swarts et al. / Biochimica et Biophysica Acta 1247 (1995) 215-224

ponents T y r and Fe(III) 2. The acid dissociation processes for acetohydroxamic acid and benzohydroxamic acid to give CHaCONHO- and C6HsCONHO- respectively, p K a values of 8.9 and 9.4 [38,39], are out of range of the present studies, and no effect of pH was observed in the case of these two reductants. The stability of R2 is less at pH > 8.6 and it was not possible to further extend the pH range. There is at present only limited information regarding the reduction potentials ( E ~' vs. NHE) of the Tyr" and Fe(III) 2 components of the active centre of R2. Unlike the studies reported for redox interconversions of the Fe(III) 2 site of hemerythrin [47,48], no evidence has been obtained in this work for any build-up of the Fe(II)Fe(III) state of R2, and a single stage conversion to Fe(II) 2 is observed. This suggests that E1~ for the reduction of Fe(III) 2 is more negative than E2~' for reduction of Fe(II)Fe(III), resulting in a rapid second stage of reduction. From observations as to whether long-lived organic radicals (e.g., that obtained from methyl viologen) reduce the Fe(III) 2 component of R2, E1~ has been estimated to be in the range - 1 1 0 mV to + 11 mV at pH 7.5, [46]. Consistent with these observations Stankovich and colleagues have reported a value - 115 mV using an electrochemical method [49]. From studies on free tyrosine and tyrosine-containing small peptides, E ~ values for the Tyr'-TyrH couple in the range 780-940 mV have been reported [50,51]. The value of 940 mV obtained at pH 7 decreases at higher pH values as the phenolate anion is formed. Since theTyr-122 radical of R2 is in a hydrophobic pocket and 10 A buried, its E'* value may be substantially modified. In the present studies the Tyr' is reduced in preference (or before) the Fe(III) 2, consistent with a reduction potential for the Tyr" couple which is more positive than that for Fe(III) 2. In the R2 reactivation process 02 is known to bind to the Fe(II) 2 [52,53], and similarly azide as N 3 is able to complex with the Fe(II) 2 [54]. There is a need therefore to consider the possibility of penetration of the active centre in the case of reagents used in the present studies. If penetration occurs to gain a closer approach to the Tyr', then H-atom transfer as in (12) R2-Tyr" + RNHOH ~ R2-TyrH + RNHO"

(12)

becomes a possibility which has to be considered rather than ET. Electrochemical studies on the reductants have yielded mid-point redox potentials. However, rate constants kl, Table 3, do not correlate well with these values. The aromatic containing reagents benzohydroxamic acid, catechol and Didox for example appear to be the stronger reductants, but with the exception of catechol react more slowly with the Tyr' of R2. The similar reaetivities of the two smaller reagents hydroxyurea and N-methylhydroxylamine at high pH are noted. If size and mid-point potential are the only important feature then acetohydroxamic acid might have been expected to give a similar rate constant.

While some results are consistent with penetration of the Tyr" site such a mechanism is not able to explain all aspects of the reactivity. In the case of catechol and Didox, since Fe(III) is not spontaneously released from metR2, then a mechanism of catechol/Didox induced release of Fe(III) has to be invoked, and penetration of the metR2 active centre to access the Fe(III) is a possible explanation. Amino-acid sequences have been determined for ten R2 subunits, and only 16 residues are conserved within all species [12,55]. The upper surface of the R2 heart shape is considered as the interaction area because it contains the only invariant surface residues and because the important (for reactivity) flexible C-terminus peptide of R2 is on this side of the heart. The latter surface is therefore a focus of attention in considering the reactivity of R2. As far as penetration is concerned the X-ray crystal structure of R2 has provided no information regarding the existence of a channel or crevice from the surface to the Tyr" [12]. To test for penetration the reactivity of four amino-acid substituted hydroxamic acid derivatives of different chain length were therefore included in this study. All the reactions were found to be slow, Table 3. In the case of the L-histidine, L-lysine and glycine derivatives the NH~ component is four bonds removed from the OH of the hydroxamic acid group. In the case of the L-glutamic acid derivative, the same NH~ group is six bonds removed from the hydroxamic acid. If charge on the reductant is excluded from penetrating the protein at the upper surface, then reagents with the longer hydrocarbon chain might gain access and approach the buried active centre more closely. This implies that the amino acid with the longest chain length should react the fastest. The order of reactivities supports this mechanism, the L-glutamic acid derivative reacting 8-10-times faster than the other three derivatives, Table 3. The maximum stretch distances from the H of the hydroxamic acid OH group to half the NH~--C bond, usin~ standard bond lengths and angles [56], range from 5.6 A (glycine) to 8.1 A (L-glutamic acid), are less than the 10 A required for the Tyr' to contact the phenolate O-atom. They are, however, within range for H-atom transfer to occur. Saturation kinetic behaviour is observed and has been quantified in the case of the L-glutamic acid derivative, but K is small and only 5.6 M-1 for association prior to reduction. While these results are consistent with penetration by a single access route, the range of compounds and the rate constants are too small for the studies to be further extended and for definitive conclusions to be made. The observation that the L-glutamate derivative (96 mM) > 50% blocks the reaction of R2 with hydroxyurea (16 mM), indicates that these two reagents react at the same region on the R2 surface. The observation that with hydroxyurea saturation kinetics are not observed ( K < 2 M -1) is consistent with an earlier report [32]. However, the step involving prior association may not be established if subsequent stages of the reaction are rapid. Alternatively there may be no single

J.C. Swarts et al. / Biochimica et Biophysica Acta 1247 (1995) 215-224

entry site/path from the surface and therefore no single unique process involving blocking. Instead protein dynamics may play an important role, where a number of different routes for penetration can be relevant. Such a mechanism has been considered in the case of CO-binding molecules such as myoglobin [57,58]. Alternatively, a mechanism of electron transfer (ET) reduction of the active centre of R2 has been proposed from Trp-48, on the upper surface of the heart shape, to Asp-237, His-ll8, Fel, and then via Asp-84 to Tyr-122. The first part of this pathway is the same as that proposed for ET between cytochrome-c peroxidase and cytochrome c [59]. However, in the present studies the observation that reduction is almost exclusively at the more readily reduced Tyr" suggests that an ET route via Fel may not be energetically favourable. With other reagents in which both the Tyr" and Fe(III) 2 are reduced, [42,46] as indeed with N-methylhydroxylamine in the present study, an important observation is that the two stages of reaction overlap, indicating rate constants which are numerically not too different and within an order of magnitude of each other. This suggests that the reduction potentials for the T y r and Fe(III) 2 are not widely separated, or that the Fe site is more accessible in certain circumstances, which might be that pertaining when Fel is involved in the pathway for ET to Tyr'. However it then becomes difficult to understand why in the present studies reduction of T y r alone should be relevant unless a different mechanism involving penetration with H-atom transfer rather than ET is observed.

Acknowledgements We thank the UK Science and Engineering Research Council for a Research Grant and the University of the Orange Free State in Bloemfontein for sabbatical leave (J.C.S.), the Nato and Natural Science and Engineering Research Council of Canada for a post-doctoral fellowship (M.A.S.A.), the Croucher Foundation for a scholarship (K.-Y.L.) and the North of England Cancer Research Campaign and the British Council (J.-Y.H.) for financial support. We are also most grateful to Mr. Kim Robson for his valuable assistance with microbiological procedures and techniques. We are particularly indebted to Professor Britt-Marie Sj6berg for most helpful advice and encouragement during the course of this work.

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