(Received for publication, October 27, 1995; and in revised form, January 9, 1996)
From the
The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an
oxygen-sensitive organic radical, which is essential for catalysis. The
radical was tentatively proposed to be on glycine 681, based on a
comparison with the glycyl radical-containing enzyme pyruvate
formate-lyase. By EPR spectroscopy of selectively H- and
C-labeled anaerobic ribonucleotide reductase, the radical
was now unambiguously assigned to carbon-2 of a glycine residue. The
large
H hyperfine splitting (1.4 millitesla) was assigned
to the
-proton. Site-directed mutagenesis was used to change
glycine 681 into an alanine residue. In separate experiments, the two
adjacent residues, cysteine 680 and tyrosine 682, were changed into
serine and phenylalanine, respectively. All mutated proteins were
retained on dATP-Sepharose, indicating that the mutant proteins had
intact allosteric sites. They also contained amounts of iron comparable
with the wild type reductase and showed the same iron-sulfur-related
spectrum, suggesting that the mutant proteins were properly folded. Of
the three mutant proteins only the G681A protein completely lacked the
detectable glycyl radical as well as enzyme activity. Our results
identify glycine 681 as the stable free radical site in E. coli anaerobic ribonucleotide reductase.
Ribonucleotide reductases are essential enzymes in all living organisms. They catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides by radical chemistry. Three different reductases have been found in Escherichia coli, one functional during aerobic growth (for a recent review, see Sjöberg(1994)) and one during anaerobic growth (Reichard, 1993b), whereas the third one is cryptic during normal growth (Jordan et al., 1994). Both aerobic and anaerobic reductases are free radical-containing enzymes, but they utilize different mechanisms for radical generation (Reichard, 1993a; Sjöberg, 1995).
The anaerobic ribonucleotide
reductase, which is encoded by the nrdD gene (Sun et al., 1993), is a homodimer of 160 kDa. This active protein contains a
stable organic free radical and a poorly defined iron-sulfur cluster
(Mulliez et al., 1993). The radical is introduced into an
inactive form of the reductase in a reaction requiring a 17.5-kDa
protein (Sun et al., 1995), S-adenosylmethionine
(AdoMet), NADPH (Eliasson et al., 1990; Harder et al., 1992), flavodoxin, and ferredoxin (flavodoxin) NADP oxidoreductase (abbreviated flavodoxin reductase below) (Bianchi et al., 1993a, 1993b). The organic radical is
oxygen-sensitive, and exposure of the radical-containing enzyme to air
leads to truncation at Gly-681 (King and Reichard, 1995), accompanied
by inactivation (Sun et al., 1993). There is a striking amino
acid sequence similarity between a stretch of 5 residues comprising
glycine 681 in the anaerobic reductase (Sun et al., 1993) or
glycine 580 in the corresponding bacteriophage T4 anaerobic
ribonucleotide reductase (Young et al., 1994) and the sequence
surrounding glycine 734 at the active site of the glycyl
radical-containing enzyme pyruvate formate-lyase (PFL) (
)(Wagner et al., 1992). Formation of an
oxygen-sensitive radical at Gly-734 in PFL and oxygen-dependent
truncation of PFL at this position are analogous reactions to the ones
described above for the anaerobic reductase. Glycine 681 in the
anaerobic reductase was therefore proposed to be the position of the
free radical in this system (Sun et al., 1993). In agreement
with this proposition, the EPR spectrum of the radical of the anaerobic
reductase shares some common features with that of the glycyl radical
of PFL, in particular the large dominant doublet hyperfine splitting
(Mulliez et al., 1993).
In this study, the chemical nature
of the free radical of the anaerobic ribonucleotide reductase has been
investigated by isotopic substitution experiments. The effects of
isotopically labeling the glycines of the enzyme on the hyperfine
structure of the characteristic EPR spectrum were consistent with the
organic radical being on a glycine residue. Moreover we used
site-directed mutagenesis to identify the location of the glycyl
radical. In addition to the mutation G681A, the adjacent mutations
C680S and Y682F were also constructed. Mutant proteins were
characterized with regard to the presence of iron-sulfur cluster,
glycyl radical, and enzyme activity. Taken together our results
demonstrate that the free radical of anaerobic ribonucleotide reductase
is located on glycine 681. In a similar study, the stable glycyl
radical of the T4 anaerobic ribonucleotide reductase was localized to
position 580 in this enzyme. ()
The E. coli anaerobic ribonucleotide reductase gene, nrdD, has previously been cloned in different plasmids for overproduction of the enzyme. The downstream nrdG gene encodes a 17.5-kDa protein, which is essential for activation of the anaerobic ribonucleotide reductase (Sun et al., 1995). Only the enzyme purified from constructs containing both nrdD and nrdG genes is active, and plasmid pREH (Sun et al., 1995), used for the isotopic labeling experiments, is one such plasmid. In this study, we also constructed plasmid pDA containing both nrdD and nrdG genes. Bacteria carrying plasmid pDA with a wild type or mutant nrdD gene gave good overexpression; extracts contained 6-12% anaerobic ribonucleotide reductase (data not shown).
Figure 1:
EPR spectra of wild type and
isotopically labeled anaerobic ribonucleotide reductase. X-band EPR
spectra of the unlabeled wild type enzyme (Spectrum A),
[2-C]glycine-labeled enzyme (Spectrum
B), and [
H]glycine-labeled enzyme (Spectrum C) are shown. The enzyme preparations were from
JM109(DE3)/pREH cells grown in the presence of 6 mM unlabeled
and labeled glycine, respectively. Spectra were recorded at 100 K;
microwave power, 50 microwatts; and modulation amplitude, 1.6
G.
The glycyl radical of PFL has the same coupling
characteristics with a large H hyperfine splitting (15 G)
and an estimated A
hyperfine coupling constant
also in the range of 16-21 G. A theoretical model study of a
dipeptide analog of a glycine radical demonstrated that only planar or
nearly planar conformations are energetically accessible due to
-electron delocalization. The hyperfine couplings computed for
these conformations (Barone et al., 1995) are in excellent
agreement with the experimental values for both glycyl radicals. On the
other hand, it is possible to distinguish between the glycyl radicals
of the anaerobic reductase and PFL. First, over a 24-h incubation
period (Mulliez et al., 1993) (
)the reductase
radical does not exchange its H
atom, responsible for the doublet
splitting, with the solvent, whereas the PFL radical exchanges rapidly
under these conditions. Second, the EPR spectrum of the PFL radical is
more complex, with partially resolved subdoublet splitting, arising
from two nonexchangeable protons (Wagner et al., 1992).
The EPR signal of the glycyl free radical also can be recorded directly in bacterial pellets from E. coli JM109(DE3)/pREH cells induced for overexpressing the reductase. The EPR spectra of cells grown in minimal medium in the presence of normal or labeled glycine gave EPR spectra identical to those of the corresponding pure enzyme. This shows that the reductase exists in the radical form within anaerobic cells and that its concentration is such that the endogenous PFL signal is not detectable. On the other hand, in plasmid-free cells only the background glycyl radical signal typical of PFL (Knappe and Sawers, 1990) was observed.
The iron-sulfur center of the anaerobic ribonucleotide reductase is essential for enzyme activity, which was found to correlate linearly with the iron content of the protein (Mulliez et al., 1993). The iron content of the protein preparations purified from the mutants varied between 1.1 and 1.3 atoms of iron/protein dimer (Table 1). These values are similar to the iron content of wild type protein preparations from JM109(DE3)/pDA cells, which is typically between 1.2 and 1.5 iron atoms. The presence of intact iron-sulfur centers in the preparations from the mutants was verified by UV-visible spectroscopy. Fig. 2shows that mutant and wild type preparations display a spectrum between 300 and 600 nm, characteristic of iron-sulfur proteins. The small difference between the spectra in the region between 300 and 350 nm is not considered to be significant. The absorption coefficients at 420 nm for the mutant proteins are similar and compare well with those published for the active enzyme prepared from plasmid pEH10 (Mulliez et al., 1993).
Figure 2: UV-visible absorption spectra of wild type and mutant anaerobic ribonucleotide reductases. A, native protein; B, G681A; C, C680S; D, Y682F. The enzymes were in 30 mM Tris-HCl, pH 7.5, 20 mM sodium formate, and 1 mM ATP. For clarity the different traces have been offset by 0.05 absorbance unit relative to each other.
If the catalytically essential radical of anaerobic ribonucleotide reductase is located at glycine 681, we would expect the mutant G681A to be devoid of both radical and enzyme activity. Fig. 3shows the EPR signals recorded from the activated proteins. With the exception of the G681A, which completely lacked a radical EPR signal, all the other proteins showed the typical glycyl radical signal. We also quantified the amount of radical (Table 1). In the wild type protein the radical content is between 0.43 and 0.49 radical/reductase enzyme. The mutant protein C680S has about 20% radical and Y682F has about 12% radical/reductase, compared with the wild type protein (Table 1). Since all purified enzymes contained substantial amounts of truncated polypeptides (see below), the actual radical content per full-length protein might be significantly higher.
Figure 3: EPR spectra of wild type and mutant anaerobic ribonucleotide reductases. The EPR spectra were recorded from activated proteins at 10 K under nonsaturating microwave power conditions. The protein concentration of the wild type is 31.5 µM. C680S is 27.8 µM, Y682F is 26.6 µM, and G681A is 29.8 µM. The EPR spectra correspond to radical concentrations of 15.5, 4, 1.8, and 0 µM, respectively. Spectra were recorded with microwave power of 20 microwatts; modulation amplitude is 3.0 G. Relative gains x1 and x4 are indicated.
The EPR signal of the glycyl radical also can
be recorded directly in bacterial pellets from induced JM109(DE3)/pDA
cells, thereby avoiding extensive purification protocols, which may
affect the recovery of the radical. Whereas cells carrying the wild
type plasmid gave the glycyl radical signal typical of anaerobic
ribonucleotide reductase, cells carrying the G681A plasmid only showed
the glycyl radical signal from PFL (data not shown), as expected if the
induced mutant anaerobic reductase lacks a glycyl radical. In this
case, washing of the cell pellet with anaerobic DO resulted
in reduced line width of the EPR spectrum (data not shown), in
agreement with the solvent exchangeability of the proton responsible
for the dominating doublet hyperfine splitting of the PFL radical.
Enzyme activities of the pure wild type and mutant reductases are shown in Table 1. The G681A mutant protein completely lacked enzyme activity, whereas the C680S protein had about 14% activity and the Y682F protein about 7% as compared with the wild type protein. Taken together, these results clearly show that the G681A mutant protein lacks both radical and enzyme activity, whereas the C680S and Y682F mutant proteins still retain some radical and some enzyme activity. The difference between radical content and corresponding enzyme activity of the mutants C680S and Y682F might be caused by experimental variation or by conformational changes introduced by the mutations that influence the enzyme activity and the generation of radical differently.
What are the functions of Cys-680 and Tyr-682?
Our results indicate that their presence influences the surroundings of
the radical-harboring Gly-681 residue but that neither is essential for
formation of the glycyl radical or for activity of the anaerobic
reductase. The mutation C680S generates the sequence RVSGY between
residues 678 and 682, which in fact is identical to the corresponding
sequence surrounding the glycyl radical of PFL (Frey et al.,
1994; Sun et al., 1993). In reciprocal experiments in the PFL
system, substitution of Ser by Cys in the active site oligopeptide
RVSGYLG led to loss of substrate efficiency (Frey et al.,
1994), whereas substitution of Ser by Ala in PFL proper gave an enzyme
with 34% residual activity (Frey et al., 1994). ()In addition, substitution of Tyr by Phe in the PFL active
site oligopeptide only led to partial loss of substrate efficiency.
Thus, mutations of the residues adjacent to the essential glycine
residue are tolerated to some extent both by the anaerobic reductase
and by PFL.