It was observed that a decrease or increase in the amino acid volume resulted in a decrease in the catalytic efficiency of the enzyme without altering the protein structure.. A strong int
Trang 1ferredoxin-NADP(H) reductase by the amino acid
volume around the catalytic site
Matı´as A Musumeci, Adria´n K Arakaki, Daniela V Rial, Daniela L Catalano-Dupuy and
Eduardo A Ceccarelli
Molecular Biology Division, Instituto de Biologı´a Molecular y Celular de Rosario (IBR), Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Argentina
Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs,
EC 1.18.1.2) are a widely distributed class of
flavoen-zymes that have non-covalently bound FAD cofactor
as a redox center FNRs participate in a wide variety
of redox-based metabolic reactions, transferring
elec-trons between obligatory one- and two-electron
carri-ers and therefore functioning as a general electron
splitter In non-phototrophic bacteria and eukaryotes,
the reaction is driven towards ferredoxin (Fd)
reduc-tion, providing reducing power for multiple metabolic
pathways, including steroid hydroxylation in
mamma-lian mitochondria, nitrite reduction and glutamate
synthesis in heterotrophic tissues of vascular plants,
radical propagation and scavenging in prokaryotes, and hydrogen and nitrogen fixation in anaerobes (for
a review, see [1,2]) In plants, FNR participates in photosynthetic electron transport, reducing Fd at the level of photosystem I, and transferring electrons to NADP+ This process ends with the formation of the NADPH necessary for CO2 fixation and other biosyn-thetic pathways [2]
The three-dimensional structures of several FNRs have been determined They display similar structural features, which have been defined as the prototype for
a large family of flavoenzymes [3–10] Plant-type FNRs can be classified into a plastidic class, characterized by
Keywords
catalytic efficiency; enzyme evolution;
ferredoxin; ferredoxin-NADP(H) reductase;
oxidoreductases
Correspondence
E A Ceccarelli, Molecular Biology Division,
Instituto de Biologı´a Molecular y Celular de
Rosario (IBR), CONICET, Facultad de
Ciencias Bioquı´micas y Farmace´uticas,
Universidad Nacional de Rosario, Suipacha
531, S2002LRK Rosario, Argentina
Fax: +54 341 4390465
Tel: +54 341 4351235
E-mail: ceccarelli@ibr.gov.ar
(Received 1 November 2007, revised 8
January 2008, accepted 16 January 2008)
doi:10.1111/j.1742-4658.2008.06298.x
Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in plastids, mitochondria and bacteria Plastidic FNRs are quite efficient reductases In contrast, FNRs from organisms possessing a heterotrophic metabolism or anoxygenic photosynthesis display turnover numbers 20- to 100-fold lower than those of their plastidic and cyanobacterial counterparts Several structural features of these enzymes have yet to be explained The residue Y308 in pea FNR is stacked nearly parallel to the re-face of the fla-vin and is highly conserved amongst members of the family By computing the relative free energy for the lumiflavin–phenol pair at different angles with the relative position found for Y308 in pea FNR, it can be concluded that this amino acid is constrained against the isoalloxazine This effect is probably caused by amino acids C266 and L268, which face the other side
of this tyrosine Simple and double FNR mutants of these amino acids were obtained and characterized It was observed that a decrease or increase in the amino acid volume resulted in a decrease in the catalytic efficiency of the enzyme without altering the protein structure Our results provide exper-imental evidence that the volume of these amino acids participates in the fine-tuning of the catalytic efficiency of the enzyme
Abbreviations
Fd, ferredoxin; Fld, flavodoxin; FNR, ferredoxin (flavodoxin)-NADP(H) reductase; IPTG, isopropyl thio-b- D -galactoside.
Trang 2an extended FAD conformation and high catalytic
efficiency (turnover numbers in the range 100–
600 s)1), and a bacterial class displaying a folded
FAD molecule and very low turnover rates (2–27 s)1)
[2,11] The Km values for NADP(H), Fd and
flavo-doxin (Fld) remain in the low micromolar range for
all reductases [2]
Two tyrosine residues interact with each side of the
isoalloxazine in plastidic FNRs On the si-face of the
flavin, which is buried within the protein structure, a
tyrosine aromatic side-chain (Y89 in pea FNR) makes
angles between 54 and 64 with the isoalloxazine in a
conformation that is at the energy minimum (Fig 1A)
[12] This residue participates in an intricate network
of interactions that involve other amino acids and the
prosthetic group, contributing to the correct
position-ing of FAD and the substrate NADP+[12] The other
tyrosine (Y308 in pea FNR) is conserved in all
plant-type plastidic FNRs stacked coplanar to the re-face of
the isoalloxazine moiety and interacts extensively with
it (Fig 1A) ([1,2,13] and references therein) This
tyro-sine has been implicated in catalysis, modulation of
the FAD reduction potential, inter- and intra-protein
electron transfer processes [14–18] and determination
of the specificity and high catalytic efficiency [15,17–
19] Using NMR techniques, it has been shown that
the maize FNR homolog Y314 is perturbed on
NADP+binding, as is the carboxyl terminal region of
the protein [20] Recently, experimental evidence for
the mobility of the carboxyl terminal backbone region
of FNR and, mainly, Y308 has been provided [19],
indicating that this movement is essential for obtaining
an FNR enzyme with high catalytic efficiency
During catalysis, the nicotinamide ring must move
to the re-face of the isoalloxazine moiety for electron transfer to occur Thus, Bruns and Karplus [3] have proposed that the aromatic side-chain of the carboxyl terminal tyrosine should be displaced to allow the sub-strate to move into the correct position (named ‘in’ conformation) The interaction of the phenol ring of Y308 with the isoalloxazine should be precisely adjusted to facilitate the ‘in’ and ‘out’ conformations
of the NADP(H) nicotinamide A strong interaction of Y308 with the flavin would impede the ability of nico-tinamide to go into the site; meanwhile, a slight inter-action would favor the stacking of the nicotinamide onto the isoalloxazine, thus decreasing the turnover rate of the enzyme, as previously demonstrated with mutant FNRs lacking this amino acid [15,17,21]
By computing ab initio molecular orbital calcula-tions, the geometry of the tyrosine and flavin has been analyzed It is proposed that Y308 is constrained against the isoalloxazine in a forced conformational arrangement This arrangement could be a consequence
of the influence of amino acids C266 and L268, which face the other side of this tyrosine (see Fig 1A), forcing
it to adopt a more planar orientation with respect to the flavin C266 is conserved between all FNRs and FNR-like proteins Homologous residues to L268 are found in the reductases from plant leaves, plant roots, cyanobacteria (blue–green algae) and all algal groups (Chlorophyta, Rhodophyta and Glaucocystophyta)
Fig 1 Computer model showing the flavin and Y308 arrangement in FNR (A) FAD cofactor, Y308 stacked on the re-face of the flavin and amino acids C266, G267 and L268 flanking Y308, as found in pea FNR (B) Computer graphic based on X-ray diffraction data for pea FNR [21], with the 266–270 loop, FAD prosthetic group and the terminal Y308 shown in dark grey.
Trang 3Leu268 is replaced by a serine in the bacterial
reducta-ses of subclass I (Azotobacter vinelandii) and by an
asparagine in the bacterial reductases of subclass II
(Escherichia coli) [22] The equivalent residues to L268
in other FNR-like enzymes are less conserved, being
proline, aspartic acid, serine or alanine
Simple and double FNR mutants of amino acids
C266 and L268 were obtained and characterized It
was observed that alteration of the amino acid volume
decreases the catalytic efficiency, suggesting that these
steric considerations may be a requirement for high
catalytic efficiency The mutations did not produce a
significant perturbation of the overall protein structure
and did not affect the oxidase activity of the
flavo-enzyme Our results suggest that these amino acids
participate in the fine-tuning of enzyme efficiency,
modulating the interaction of Y308 and⁄ or the
nicotin-amide with the isoalloxazine This type of modulation
of aromatic residue interactions could be a general
strategy occurring in enzyme structures
Results
Ab initio molecular orbital calculations
The geometries of aromatic amino acids facing the
re-face of the flavin were determined using
high-resolu-tion plant-type FNR crystal structures It was observed
that these tyrosines always interact in face-to-face
posi-tions (Fig 1A; Table 1) The B ring of the flavin is
always involved in this interaction in a nearly parallel
position in which the angle formed with the tyrosine
phenol and isoalloxazine varies from 0 to 6 in all
high-efficiency plastidic FNRs and 15 for the
ferre-doxin-NADP(H) reductase from E coli To gain a better understanding of this interaction, the geometric preferences of the above-mentioned interaction were analyzed using model molecules and ab initio mole-cular orbital calculations with the restricted Hartree Fock theory level and a 6-311 + G(d,p) basis set A simplified system was constructed containing lumiflavin (7,8,10-trimethylisoalloxazine), which is an accepted flavin model compound for calculations [23], and phe-nol as the tyrosine R group This system has been used previously to analyze the geometry of the tyrosine stacked on the si-face of the flavin in FNRs [12] The single point energies of the flavin–tyrosine system in different conformations were calculated The arrange-ment of lumiflavin and phenol with the exact geometry found between flavin and Y308 in the crystal structure
of pea FNR was used for the initial set-up Then, dif-ferent arrangements were generated in which the phe-nol placed in this exact position was rotated around the Cc–Cf axis in discrete steps, keeping the orienta-tion of the phenol hydroxyl group and the distance between the aromatic ring centroids constant (see Fig 2A) This allowed us to obtain arrangements of the two molecules with angles (a) from)75 to 90 Figure 2B illustrates the differences in potential energy values determined for arrangements of the phe-nol–lumiflavin pair plotted against the angle a, as depicted in Fig 2A, at a centroid distance of 3.6 or 4.6 A˚ The value obtained for the natural geometry of the carboxyl terminal tyrosine in pea FNR (5.8) was used as reference These distances were chosen consid-ering the tyrosine–flavin arrangement found in FNRs and because energetically favorable, non-bonded, aro-matic interactions occur in proteins at phenyl ring centroid separations of > 3.4 and < 7 A˚ [24]
A global energy minimum was theoretically detected between 11 and 22 at a distance between centroids of 3.6 A˚ The angle found in E coli FNR was the closest
to the minimum of the plot In all plastidic FNRs, the position of the tyrosine was near the minimum (repre-sented in Fig 2B with open circles and a number indi-cating the enzyme) Any position that does not fall within)10 to 37 notoriously decreases the stability of the pair, increasing repulsion, probably as result of steric constraints between the two aromatic rings When the total energy of the system was analyzed at a centroid– centroid distance of 4.6 A˚, a minimum was observed at 40 and a shallow low-energy region was detected from 20 to 55 Moreover, all differences in potential energy values obtained for arrangements at 4.6 A˚ between angles from )20 to 85 were equal or lower than the energy calculated for the observed arrangements found
in plastidic FNR enzymes in nature (Fig 2B) All FNRs
Table 1 Angles and distances between the tyrosine interacting
with the re-face of the flavin and the isoalloxazine B ring obtained
from FNR crystal structures.
FNR source Type
Maximal angle (deg) a
Centroid distance (A ˚ ) b
PDB
ID Reference Paprika Plastidic 5.09 3.70 1sm4 [6]
Spinach Plastidic 0.01 3.65 1fnb [3]
Anabaena Plastidic 5.40 3.60 1que [4]
Synechococcus
sp.
Plastidic 0.40 3.50 2b5o Unpublished
E coli Bacterial 15.00 3.60 1fdr [10]
a Angle formed between the tyrosine and the re-face of
isoalloxa-zine, measured as shown in Fig 2A b Distance (d ) from the center
of the phenol ring to the center of the proximal flavin ring, as
shown in Fig 2A.
Trang 4displayed geometries for the re-face tyrosine phenol and
flavin falling near or into the minimum energy valley
with a centroid separation of about 3.6 A˚ However, if
the tyrosine were able to move away from the flavin, a
more stable arrangement was possible between both
aromatic rings, allowing them to gain up to
approxi-mately 5.8 kcalÆmol)1 of stabilization energy, as
calcu-lated from Fig 2B Thus, it may be inferred that the
position of the re-face tyrosine in FNRs is not governed
by the energetic minimum of the pairwise flavin–phenol
interaction By analyzing the crystal structure of pea
FNR, it was deduced that Y308 is constrained against
the isoalloxazine in an unfavorable conformational
arrangement by the influence of amino acids C266 and
L268 These residues face the other side of this tyrosine
and are members of a conserved loop (266CGLKG270)
that shapes part of the FNR catalytic site (see
Fig 1A,B) They may force Y308 to adopt a more
pla-nar orientation with respect to the flavin The overall
result is a less stable conformational arrangement
Design and construction of C266, G267 and L268
single and double FNR mutants
Five single mutants of C266, G267 and L268 and a
double mutant of C266 and L268 were successfully
constructed and confirmed by DNA sequencing The
design of the mutants was intended to preserve the
amino acid character and to modify only the relative
volume of their R groups
The expression of the FNR mutants as soluble
cyto-solic proteins in E coli was analyzed using SDS-PAGE
and western blot (not shown) The expression levels of
FNR mutants C266AL268A, C266A and L268V were similar to those of recombinant wild-type FNR These recombinant enzymes were largely recovered in the sol-uble fraction after the induction of protein expression
at 25C, disruption of E coli cells and fractionation
by centrifugation In contrast, replacement of either G267 with a valine or C266 with a leucine or methio-nine produced a notorious precipitation of the expressed polypeptide FNR mutants C266L and C266M were successfully expressed at 15 C during
16 h with 0.1 mm isopropyl thio-b-d-galactoside (IPTG) Both C266L and C266M mutant enzymes showed a higher FAD release rate [4.8· 10)2 and 2.6· 10)2lmolÆFADÆh)1Æ(lmolÆFNR))1, respectively] than the wild-type enzyme [1.1· 10)5lmolÆFADÆ
h)1Æ(lmolÆFNR))1], as determined by measuring the increase in FAD fluorescence [22] after incubating the enzymes for 5 h at 25C These observations suggest a weaker FAD interaction with the apoprotein, and may explain the difficulties in obtaining these enzymes in soluble form during protein expression in E coli Attempts to purify mutant enzyme G267V were unsuc-cessful and no further analysis was possible
All reductase variants were excised from the carrier protein using thrombin protease and, after chromato-graphy on nickel-nitrilotriacetic acid agarose, were obtained in homogeneous form as judged by SDS-PAGE (not shown)
FAD content and spectral properties Analysis of the UV–visible absorption properties of the different FNR mutants showed small changes,
Fig 2 Computed relative free energy calculations for the lumiflavin–phenol interaction (A) Scheme of the coordinate system used to define the relative positions of phenol and lumiflavin, as found for Y308 and flavin in pea FNR a, Dihedral interplanar angle between rings (for clar-ity, only three positions are shown); d, distance between ring centroids (B) Relative free energy of the arrangement shown in (A) as a func-tion of the stated a angles at fixed distances of 3.6 A ˚ ( , full line) and 4.6 A˚ ( , broken line) Open symbols indicate the observed values for the different plastidic FNRs as follows: 1, paprika; 2, spinach; 3, Anabaena; 4, pea; 5, maize; 6, Synechococcus sp.; 7, E coli Ab initio molecular orbital calculations were performed as described in Materials and methods.
Trang 5indicating slight variations in the environment of the
flavin prosthetic group All proteins displayed a typical
FNR spectrum with maxima at approximately 380 and
460 nm and shoulders at 430 and 470 nm The
absor-bance maximum of the transition band of the wild-type enzyme at 386 nm was shifted slightly to 381 nm
in the C266L, C266M and C266A mutants, but not in the L268V mutant At 459 nm, all changes were within the detected error (Fig 3A) These shifts may indicate modification of the isoalloxazine environment, although none of the amino acids that directly interact with the flavin were modified The FAD content of the wild-type and mutant enzymes was determined by release in the presence of 0.2% SDS [25] FAD : poly-peptide stoichiometry values of 0.85–0.99 were calcu-lated for all mutants (not shown) Therefore, the amino acid changes introduced do not prevent assem-bly of the prosthetic group and do not impede the pro-duction of a folded protein, although, as mentioned above, they may affect the protein folding process
CD spectra were recorded for wild-type and mutant FNRs in an effort to assess the impact of the amino acid changes on the structural integrity of the reducta-ses Wild-type and mutant FNRs had very similar spectra, exhibiting a negative region from 204 to
240 nm, with a minimum similar for all proteins, and
a positive ellipticity at 202 nm (Fig 3B) The near-UV and visible CD spectra (Fig 3C) of the proteins were also very similar, showing the typical spectrum for FNR [26], with positive ellipticity in the region of the first flavin visible absorption band, and with a peak at approximately 380 nm for the wild-type enzyme and
370 nm for the mutant proteins This is consistent with the alteration observed in the absorbance spectra of the mutants A less intense band of negative ellipticity was observed in the region of the second flavin visible band at 470 nm for the wild-type enzyme and mutant proteins (Fig 3C) In the near-UV region, all FNRs exhibited very strong, sharp positive and negative sig-nals at 271 and 286 nm, respectively A similar strong signal at 272 nm, observed in the CD spectrum of
E coli Fld oxidoreductase, has been attributed to the stacked interactions between FAD and one or more aromatic residues [27] The introduced mutations in FNR did not alter the position of this near-UV band Some changes in intensity were observed in the FNR mutants, indicating some perturbation of the symmetry relationships between the isoalloxazine chromophore
Fig 3 Absorbance and CD spectra of wild-type and mutant FNRs Absorbance (A) and CD (B, C) spectra of wild-type FNR (thick line), L268V (thin line), C266AL268A (thick dotted line), C266A (thin dot-ted line), C266L (thick broken line) and C266M (thin broken line) For spectra at 200–250 nm (B), the optical path length was 0.2 cm and the protein concentration of FNRs was 0.5 l M For spectra at 250–600 nm (C), the optical path length was 1 cm and the protein concentration of FNRs was 5 l M
Trang 6and either the carboxyl terminal tyrosine side-chain or
the surrounding protein environment Together, these
results clearly indicate that these mutations introduced
only local changes in the flavin microenvironment
Interaction with substrates and steady-state
kinetics
The alterations in the flavin absorption spectrum and
the intrinsic FAD fluorescence were used as described
previously [19,25,28] to determine the binding
con-stants for the FNR–NADP+ complexes The
differen-tial spectral changes obtained by incubation of the
wild-type and mutant enzymes with NADP+ are
shown in Fig 4 NADP+ binding to the L268V
mutant provoked spectral changes similar in shape and
intensity to that of the wild-type enzyme, with the
recognized maximum at 510 nm (Fig 4) In contrast,
mutants C266L, C266AL268A, C266A and C266M
showed progressive changes in shape and maxima of
the differential spectra, indicating a modification in the
way in which the nucleotide interacts with the flavin
and⁄ or its environment Unexpectedly, dissociation
constants for NADP+ were not significantly affected
in any of the mutants for either NADP+ or Fd
(Tables 2 and 3, respectively) The only exception was
the double mutant, which showed an increase in the
Kd value for the enzyme–NADP+ complex It has
been documented that the intensity of the FNR–
NADP+ differential spectrum peak at about 510 nm
correlates with the nicotinamide interaction on the
re-face of the isoalloxazine [17,25,28,29] It may be inferred from the spectral data presented that the inter-action of the NADP+ nicotinamide with the flavin is considerably disturbed, probably as a result of changes introduced by the mutations in the environment of the prosthetic group As a result of the important changes observed for each mutant in the differential spectra elicited by NADP+, it was decided to use an alterna-tive procedure to determine the affinity constant for the nucleotide The dissociation constants of the FNR–NADP+(Table 2) and FNR–Fd (Table 3) com-plexes were estimated by measuring flavin fluorescence and flavoprotein fluorescence quenching, respectively, after the addition of each substrate, as described in Materials and methods Similarly, the Kd value of the FNR–Fd complex was determined in the presence of NADP+ (Table 3) As shown in Table 2, values obtained for the binding of NADP+ are in good agreement with those determined by differential spec-troscopy In our hands and using this methodology, a significant decrease (7.6-fold) in the affinity of FNR for Fd was detected when NADP+was added at a sat-urating concentration, compared with the respective affinity in the absence of substrate (Table 3) Interest-ingly, in all cases, the mutations introduced diminished
or completely abolished the observed effect
The catalytic properties of the different FNR mutants were determined for two different enzymatic reactions The observed values for kcat and Km and the calculated kcat⁄ Km value for NADPH and Fd are summarized in Tables 2 and 3
L268V FNR displayed kcat and Km values for the diaphorase reaction in the region of 0.8 and 2.0 times those observed for the wild-type enzyme Similarly, the decrease in Fd was about 0.7 times that observed with the wild-type enzyme
By contrast, mutations in C266 produced a more dramatic effect on the catalytic properties of FNR Replacement of C266 with a methionine, which implies
a volume increase of 55.5 A˚3, decreased the kcat value
by more than 99.8% and increased the Km value for diaphorase activity three-fold C266AL268A FNR, in which substitutions produced an amino acid volume decrease of 96.2 A˚3, also showed a major disruption in catalytic function, with a kcat reduction of more than 99% and a 20-fold increase in Km The introduced changes resulted in a 1300- and 2200-fold decrease in the catalytic efficiency of C266M and C266AL268A, respectively
The correlation between the catalytic efficiency changes caused by the mutations and the different amino acid physicochemical properties was investi-gated All introduced mutations substituted a polar
Fig 4 Interaction of wild-type and mutant FNRs with NADP +
Dif-ferential spectra of the wild-type FNR (thick line), L268V (thin line),
C266AL268A (thick dotted line), C266A (thin dotted line), C266L
(thick broken line) and C266M (thin broken line) elicited by NADP +
binding, as obtained from the mathematical subtraction of the
absorption spectra in the absence and presence of 0.3 m M NADP +
Trang 7neutral amino acid by non-polar residues without a
change in the net charge The catalytic efficiencies of
the different enzymes were plotted as a function of the
absolute change of hydropathy according to Kyte and
Doolittle [30] (Fig 5A), the octanol–water partition
coefficient (log P) [31] and volume [32] No
correla-tions were found with changes in hydropathy (Fig 5A)
and log P (Fig 5B) In contrast, the absolute change
in volume correlated with the decrease in catalytic
effi-ciency (Fig 5C) An alteration (increase or decrease)
in volume of the amino acid at position 266 induced a
decrease in catalytic efficiency of the enzyme Mutants
with higher volume changes in this residue were more
affected The value for the reduction in catalytic
effi-ciency previously obtained by replacement of spinach
FNR C272 (homolog to pea FNR C266) with a serine
[28] was included (open symbols in Fig 5A–C and white bar in Fig 5D)
Mutations in C266 that decreased the amino acid volume resulted in a moderate increase in catalytic effi-ciency for the activity of cytochrome c reductase These results originate from the higher relative decrease in Km than kcat, with respect to the corre-sponding values observed in the wild-type enzyme, and consequently inferences from the calculated changes in catalytic efficiency may not be appropriate [33] The accumulated data reviewed by Mattevi et al [34] indicate that the rate-limiting step in the oxygen reactivity of flavoproteins is the first electron transfer step from the two-electron-reduced flavin to mole-cular oxygen In this context, the oxidase activity of the wild-type and mutant FNRs was investigated at
Table 2 Kinetic parameters for the diaphorase reaction of the wild-type (WT) and mutant FNRs, and dissociation constants for the different FNR–NADP+complexes Potassium ferricyanide reduction was measured using the diaphorase assay of Zanetti [68] in 50 m M Tris ⁄ HCl (pH 8.0).
FNR form DV a (A˚3 )
Km(NADPH) (l M ) kcat(s)1)
kcat⁄ K m
(l M )1Æs)1)
DDGMUT⁄ WTb
(kcalÆmol)1)
Kd(NADP + ) c
(l M )
Kd(NADP + ) d
(l M )
a Volume change of the R amino acid groups introduced by the mutations was determined following the standard radii and volumes calcu-lated by Tsai et al [32].bDDG MUT ⁄ WT indicates the energy barrier introduced by the mutations to the catalytic efficiency of FNR calculated
by the following equation: DDGMUT⁄ WT= )RT ln(k cat ⁄ K m )MUT⁄ (k cat ⁄ K m )WT c Determined by differential spectra using 15 l M flavoproteins
in 50 m M Tris ⁄ HCl (pH 8.0) at 25 C Absorbance differences (DA at 510 nm for the wild-type and L268V mutant FNRs, and at 390 nm for the C266A, C266AL268A, C266L and C266M mutant FNRs) were measured and plotted against increasing NADP+concentration The data were fitted to a theoretical equation for a 1 : 1 complex d Determined by fluorescence spectroscopy using oxidized flavoproteins at 8.5 l M
in 50 m M Tris ⁄ HCl (pH 8.0) at 25 C, as described in Materials and methods.
Table 3 Kinetic parameters for cytochrome c reductase of the wild-type (WT) and mutant FNRs, and dissociation constants for the com-plexes of the different FNR forms with Fd Cytochrome c reduction was followed at 550 nm (e550= 19 m M )1Æcm)1) as described in Materials
and methods ND, not determined.
FNR form
DV a
(A˚3 )
Km(Fd) (l M )
kcat (s)1)
kcat⁄ K m
(l M )1Æs)1)
K d for the FNR–Fd complex (l M )
In the presence
of NADP + (K dP ) b
In the absence
of NADP + (K dA ) b K dP ⁄ K dA
C266AL268A –96.2 0.004 ± 0.001 0.016 ± 0.0009 4.00 ± 1.22 2.74 ± 0.24 2.20 ± 0.26 1.3
a Volume change of the R amino acid groups introduced by mutations was determined following the standard radii and volumes calculated
by Tsai et al [32] b Determined by fluorescence spectroscopy using oxidized flavoproteins at 3 l M in 50 m M Tris ⁄ HCl (pH 8.0) at 25 C in the absence or presence of 0.3 m M NADP + , as described in Materials and methods.
Trang 8saturating NADPH concentration As shown in
Table 4, wild-type and mutant enzymes displayed
simi-lar oxidase activities, indicating that no changes are
evident in this process on mutation of the FNR
resi-dues under study
Thermal analysis of protein unfolding for
wild-type and mutant FNRs
Thermal denaturation determined by CD was used to
measure the stability of the FNR mutants Based on
measurements over a range of temperatures (shown in
Fig 6), parameters such as the midpoint of the
unfold-ing transition meltunfold-ing point (Tm) were calculated, and
are shown in Table 5 Curves were also analyzed on
the basis of the two-state model [35], and the
corre-sponding DSm values (entropy change at Tm) were
calculated from the slopes of DG versus T at midpoint
temperatures [35] All replacements led to less stable enzymes compared with wild-type FNR However, mutations that introduced reductions in amino acid volume caused slight to moderate changes in stability with respect to the wild-type enzyme (– 0.76 to )0.94 kcalÆmol)1) Using the foldx algorithm [36], the
C266A L268A L268V C266A C272S WT C266L C266M
–25.0 –21.5 –16.3 0.0 53.2 55.5
FNRs
ΔV (Å 3
) (absolute value)
1
0.01
0.1
10
100
0.01
0.1
1
10
100
kcat
kcat
1
Fig 5 Catalytic efficiencies of wild-type and mutant FNRs plotted as a function of different amino acid physicochemical properties The cat-alytic efficiencies of wild-type and mutant FNRs from Table 2 (percentage of the wild-type enzyme) are plotted as a function of the absolute changes in hydropathy [30] (A), octanol–water partition coefficient [31] (B), volume [32] (C) and volume change in C266 (filled bars) and L268 (hatched bar) mutants (D) The FNR mutant C272S from spinach showed a k cat ⁄ K m value five-fold lower than that of the wild-type reductase (0.40 versus 14.28 l M )1Æs–l ) [28], and is represented by an open symbol in (A), (B) and (C) and a white bar in (D) Substitution of C with S introduces a volume change of )16.3 A˚ 3
Table 4 Oxidase activity of the wild-type (WT) and mutant FNRs Oxidase activity was followed by NADPH oxidation, as described in Materials and methods.
) Oxidase activity (s)1)
Trang 9direct effect of mutations that replace native amino
acids with alanine on the overall stability of the
pro-tein was evaluated A theoretical DDG value of
)1.02 kcalÆmol)1 was obtained for the C266A mutant,
in complete agreement with our experimental results
When the amino acid mutation induced a volume
increase, important destabilizations were
experimen-tally observed C266L and C266M exhibited lower
DDG values: )8.50 and )6.80 kcalÆmol)1, respectively
These outcomes indicate that although little influence
is exerted by residue substitutions on the
destabiliza-tion of the secondary and tertiary structure (see Fig 3)
there is a considerable difference in thermal energy
change between the wild-type enzyme and mutants with replacements that increase volume
Discussion The role of the aromatic residue interacting with the re-face of the flavin in FNR-like enzymes has been analyzed, and a variety of functions have been pro-posed [14,16,18,19,37,38] In previous publications, mechanistic evidence has been presented that the inter-action of the nicotinamide of substrate NADP+ with the isoalloxazine is modulated by the terminal tyrosine (Y308 in pea FNR) [15,17,18] During binding of NADP+, the terminal tyrosine should be removed from its resting place to allow the nicotinamide to move into a productive position [21] This exchange between Y308 and the NADP+nicotinamide has been experimentally indicated as the enzyme rate-limiting step [18] Evidence has recently been presented that the mobility of the carboxyl terminal region is essential for obtaining high catalytic rates [19] Ab initio calcula-tions and mutagenesis studies were performed on the FNR enzyme with the aim of obtaining a better under-standing of the structural and functional role of this tyrosine and the interacting amino acids C266, G267 and L268 The data support the hypothesis that the aromatic interaction between the flavin, Y308 and the nicotinamide of NADP+is precisely tuned by selecting amino acids that face the other side of the tyrosine phenol ring The specific volumes of the above-men-tioned residues condition the arrangement of Y308 and the nicotinamide of NADP+in the catalytic site Non-covalent aromatic interactions are essential to protein–ligand recognition [39] Furthermore, they are widespread in biomolecules, clusters, organic⁄ biomo-lecular crystals and, more recently, in the building of nanomaterials [40] In proteins, the rings of trypto-phan, tyrosine, phenylalanine and histidine participate either in the interaction with hydrogen donors (p–H interaction) or binding with other aromatic rings (p–p interactions) [41] The latter interactions are observed
in a great variety of geometries The edge–face geome-try is commonly found between aromatic residues in proteins Other two-stacked orientations are also estab-lished, including one in which the interacting rings are offset and stacked near-planar, and arrangements of face-to-face stacked aromatic rings [42]
By analyzing the crystal structure of FNRs, it was found that the inter-ring orientational angles between the re-face aromatic ring and flavins were quite con-stant and always positioned at a limiting distance of 3.6 A˚ Our ab initio calculations indicated that Y308 in pea FNR adopts a conformation close to minimum
Table 5 Thermodynamic parameters derived from the thermally
induced unfolding curves of wild-type (WT) and mutant FNRs The
data of Fig 6 were analyzed assuming a two-state approximation
as described previously [67].
FNR form DV a (A ˚ 3 ) Tm( 0 C)
DS m
(kcalÆmol)1Ædeg)1)
DDG (kcalÆmol)1)
C266A )21.5 63.2 ± 0.3 0.71 ± 0.03 )0.94
L268V )25.0 63.5 ± 0.1 )0.40 ± 0.01 )0.76
C266AL268A )96.2 63.5 ± 0.1 0.69 ± 0.01 )0.77
C266L 53.2 50.8 ± 0.1 0.38 ± 0.01 )8.50
C266M 55.5 53.6 ± 0.2 0.43 ± 0.02 )6.80
a Volume change of the R amino acid groups introduced by the
mutations was determined following the standard radii and
vol-umes calculated by Tsai et al [32].
Fig 6 Thermal unfolding of wild-type and mutant FNRs monitored
by CD CD melting curves were recorded at 280 nm, using a
pro-tein concentration of 3 l M in 50 m M potassium phosphate (pH 8.0),
whilst the temperature of the sample was increased at a uniform
rate of 1 CÆmin)1 (from 25 to 80 C) Wild-type FNR (thick line),
L268V (thin line), C266AL268A (thick dotted line), C266A (thin
dot-ted line), C266L (thick broken line) and C266M (thin broken line) are
shown.
Trang 10energy for a distance of 3.6 A˚ However, when
calcula-tions were performed with aromatic rings stacked at
4.6 A˚, a lower energy minimum was obtained These
results suggest that, if more freedom were available for
the arrangement, the aromatic ring of the tyrosine
would adopt a T-shaped geometry, with increased
stabilization of the pair In all plastidic FNRs, Y308
homologs are close to the calculated minimum at
3.6 A˚, supporting the theoretical data obtained
More-over, it may be inferred from these observations that
the orientation of Y308 with respect to the flavin is
mainly governed by the aromatic interaction without
involvement of attractive forces from the other side of
tyrosine The relative stability of planar and T-shaped
aromatic interactions has been studied extensively, but
consolidated conclusions are still being debated The
accumulated evidence indicates that the T-shaped
structure is likely to be more stable than the planar
stacked structure, as calculated for model systems [42]
Tyrosines have been found to interact with flavins in a
myriad of arrangements, including, for example,
spa-tial T-shaped arrangements [12,34], planar parallel and
displaced stacks [1,2,13,43,44] and even near-90
T-shaped orientation [45], demonstrating that the
sur-rounding environment can condition these
arrange-ments It has been observed previously that Y89 in pea
FNR, which faces the si-face of the flavin in a
T-shaped geometry of 54, is close to the global energy
minimum [12] Similar conclusions have been found
for the phenol side-chain of the si-face tyrosine of
sev-eral FNR family flavoproteins ([12,46] and references
therein) Our calculations also indicate that the
tyro-sine–flavin bacterial arrangement in E coli FNR is
1.24 kcalÆmol)1 more stable than that observed for the
same pair in plastidic pea FNR (open circle numbered
4 in Fig 2) Thus, tyrosine displacement for
nicotin-amide binding should be easier in pea FNR than in
the bacterial enzyme As this movement was postulated
to be the rate-limiting step for catalysis [18], the
differ-ences in stability may account for the distinct turnover
numbers that are 20- to 100-fold lower for bacterial
enzymes than their plastidic and cyanobacterial
coun-terparts
Our mutants enabled the observed results to be
interpreted in terms of protein structure,
thermody-namics and function The C266 mutants are of
particu-lar interest because this residue has functional
homologs in all FNR-like structures Moreover, the
cysteine and glycine at this position are part of one of
the consensus sequences that define the structural
fam-ily [1,11] As anticipated, the final tertiary structure of
the mutants, with the exception of G267V, was
rela-tively unchanged, as shown by the fact that mutations
in FNR did not alter the near-UV band of the
CD spectra A small perturbation of isoalloxazine was detected by CD and UV–visible spectrophotometry Flavin electronic transitions in the 300–600 nm region originate from p–p transitions [26] Thus, changes in the CD spectra are expected to occur on modification
of the interaction of Y308 with the flavin Our mutants displayed variations at 370–380 nm, correlating with the changes observed in that region of the UV–visible spectra Mutations may induce either a change in the interaction strength between the flavin and Y308 or a displacement of the ‘in’ and ‘out’ equilibrium of the Y308 phenol ring [15,17,21], which could not be detected by crystal structure analysis
Alteration of the flavin environment was more noticeable when the differential spectra elicited by NADP+ binding were analyzed These changes were closely related to the magnitude of the changes intro-duced with respect to the wild-type enzyme Substitu-tion of C266 with the bulky methionine completely reverted the shape of the differential spectrum of the wild-type enzyme with NADP+, producing a profile quite similar to that already obtained for the wild-type FNR from Anabaena variabilis when the nucleotide is bound [47] The absence of the characteristic band at
510 nm for the flavin–nicotinamide interaction has been explained by the observation that the C-terminal tyrosine in this enzyme has a reduced degree of ‘out’ conformations relative to other plastidic FNRs Conse-quently, our observations may account for a reduced interaction of the nicotinamide with the flavin in the C266M mutant Moreover, spectral changes on NADP+binding to L268V are coincident with the dif-ferential spectra previously obtained for the Anabaena variabilis FNR mutant L263A [47] The Kd values obtained for NADP+ binding to the mutants were only slightly modified, with the exception of the double mutant It can be concluded that the interactions with the adenine and phosphate regions of NADP+ are conserved, and that the observed alteration is probably the result of a change in the position or extent of inter-action between the flavin and the nicotinamide Kinetic analysis of the mutants indicates that the cysteine sulfhydryl group is by no means essential for catalysis, as documented previously [28] Replacement
of C266 by any aliphatic residue produced enzymes that, even when notoriously affected in catalysis, were still active When the cysteine was substituted with a methionine, providing a sulfur atom in a nearby posi-tion, a functional enzyme was also obtained Sulfur– flavin interactions have been proposed and analyzed
by computational studies and experimental means [48,49] These studies have indicated the existence of