Biomedical subjects
Publications and source records attributed to F Cutruzzolà.
All denitrifiers can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels by controlling the expression of denitrification gene clusters by redox signalling through transcriptional regulators belonging to the CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulator) superfamily.
In denitrifying bacteria, the concentration of NO is maintained low by a tight control of the expression and activity of nitrite and NO reductases. Regulation involves redox-linked transcription factors, such as those belonging to the CRP-FNR (cAMP receptor protein-fumarate and nitrate reductase regulator) superfamily, which act as oxygen and N-oxide sensors. Given that few members of this superfamily have been characterized in detail, we have cloned, expressed and purified the dissimilative nitrate respiration regulator from Pseudomonas aeruginosa. To gain insights on the structural properties of the dissimilative nitrate respiration regulator, we have also determined the aggregation state of the purified protein and its ability to bind hydrophobic compounds such as 8-anilino-1-naphthalenesulphonic acid.
The cd(1) nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide. The enzyme contains two redox centers, a c-type heme and a unique d(1) heme, which is a dioxoisobacteriochlorin. Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d(1) cofactor with extremely high affinity and inhibit enzyme activity. In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d(1) site of the reduced enzyme. The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heme complexes. The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm(-1), respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm(-1), respectively, for the CO complex. Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heme proteins. The unusual spectroscopic characteristics of the d(1) cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand. It is likely that the influence of a highly ruffled structure of heme d(1) on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies.
Cytochrome c(551) (cyt c(551)) from Pseudomonas aeruginosa is a small protein (82 residues) that folds via a three-state pathway with the accumulation in the microsecond time-range of a compact collapsed intermediate. The presence of a single His residue, at position 16, permits the study of the refolding at pH 7.0 in the absence of miscoordination events. Here, we report on folding kinetics in the millisecond time-range as a function of urea under different pH conditions. Analysis of this process (over-and-above proline cis-trans isomerization) at pH 7.0, suggests the existence of a multiple transition state pathway in which we postulate three transition states. Taking advantage of site-directed mutagenesis we propose that the first "unfolded-like" transition state (t(1)) originates from the electrostatic properties of the collapsed state, while the second transition state (t(2)) involves the interaction between the N and C-terminal helices and is stabilized by the salt bridge between Lys10 and Glu70 ( approximately 1 kcal mol(-1)). Our results suggest that, contrary to other cytochromes c, the roll-over effect observed for cyt c(551) at low denaturant concentration can be interpreted in terms of a broad energy barrier without population of any intermediates. The third and more "native-like" transition state (M) can be associated with the breaking/formation of the Fe(3+)-Met61 bond. This strong interaction is stabilized by the hydrogen bond between Trp56 and heme propionate 17 (HP-17) as suggested by the increase in the unfolding rate at high denaturant concentration of the Trp56Phe site-directed mutant.
cd(1) nitrite reductase from Pseudomonas stutzeri is a di-haem- containing enzyme, comprising a c-type haem and a d-type haem. Studies with the highly related cd(1) nitrite reductase of Pseudomonas aeruginosa have established that this enzyme undergoes fast (microsecond) and global structural relaxation upon CO photodissociation from the reduced enzyme. A key difference between the Ps. aeruginosa and Ps. stutzeri enzyme is the absence of a flexible N-terminal extension in the Ps. stutzeri enzyme. In Ps. aeruginosa cd(1) nitrite reductase the N-terminal extension wraps around the second subunit of the homodimer and with Tyr(10) stabilizing a water molecule co-ordinated to the d(1)-haem. Given the intimate association of the N-terminal extension with the d(1)-haem, we hypothesized that the presence of the N-terminal extension likely contributes to the fast structural reorganization seen during photodissociation of CO from the reduced enzyme. In the present study we have investigated the kinetics of CO association and CO photodissociation of Ps. stutzeri cd(1) nitrite reductase (which lacks the N-terminal arm seen in the Ps. aeruginosa enzyme) to probe the role and influence of the N-terminal arm in the fast global structural reorganization seen with Ps. aeruginosa. Surprisingly, we find that Ps. stutzeri cd(1) nitrite reductase also undergoes fast structural reorganization during CO photodissociation. We also show, in stopped-flow experiments, that the kinetics of CO binding and dissociation with reduced Ps. stutzeri cd(1) nitrite reductase are similar to those observed with Ps. aeruginosa enzyme, thus ruling out a major role for the N-terminal flexible arm found in Ps. aeruginosa in the kinetics of these processes. Our data indicate that global structural reorganization following CO photodissociation is an intrinsic property of the haem domains in cd(1) nitrite reductases. The absence of an N-terminal extension, as in the Ps. stutzeri cd(1) nitrite reductase, does not lead to loss of global structural reorganization following CO photodissociation.
Apomyoglobin from Aplysia limacina (al-apoMb), despite having only 20 % sequence identity with the more commonly studied mammalian globins (m-apoMbs), properties which result in an increased number of hydrophobic contacts and a loss of most internal salt bridges, shares a number of features of their folding profiles. We show here that it contains an unusually stable core which resists unfolding even at 70 degrees C. The equilibrium intermediate (I(T)) at this high temperature is distinct from the acid unfolded state I(A) which has many properties in common with the acid intermediate observed for the mammalian apoproteins (I(AGH)). It contains a smaller amount of secondary structure (27 % alpha-helical instead of 35 %) and is more highly solvated as evidenced from its fluorescence spectrum (lambda(max)=344 nm instead of 338 nm). Its stability is greatly increased (DeltaDeltaG(w)=-6.75 kcal mol(-1)) in the presence of high salt (2 M KCl), lending support to the view that hydrophobic interactions are responsible for its stability. Kinetic data show classical two-state kinetics between I(A) and the folded state both in the presence and absence of salt. Both I(A) and I(T) can be populated within the dead time of the stopped-flow apparatus, since initiating the refolding reaction from I(T) or I(A) rather than the completely unfolded state does not affect the observed refolding time-course. Our conclusion is that al-apoMb, as other "apo" proteins (including for example alpha-lactalbumin in the absence of Ca(2+)), may be described as "uncoupled" with an unusually high and exploitable tendency to populate partially folded states.
Aplysia limacina myoglobin lacks the distal histidine (His (E7)) and displays a ligand stabilization mechanism based on Arg(E10). The double mutant Val(E7)His-Arg(E10)Thr has been prepared to engineer the role of His(E7), typical of mammalian myoglobins, in a different globin framework. The 2.0 A crystal structure of Val(E7)His-Arg(E10)Thr met-Mb mutant reveals that the His(E7) side chain points out of the distal pocket, providing an explanation for the observed failure to stabilize the Fe(II) bound oxygen in the ferrous myoglobin. Moreover, spectroscopic analysis together with kinetic data on azide binding to met-myoglobin are reported and discussed in terms of the presence of a water molecule at coordination distance from the heme iron.
We report on the folding kinetics of the small 82 residue cytochrome c551from Pseudomonas aeruginosa. The presence of two Trp residues (Trp56 and Trp77) allows the monitoring of fluorescence quenching on refolding in two different regions of the protein. A single His residue (the iron-coordinating His16) permits the study of refolding in the absence of miscoordination events. After identification of the kinetic traps (Pro isomerization and aggregation of denatured protein), overall refolding kinetics is described by two processes: (i) a burstphase collapse (faster than milliseconds) which we show to be a global event leading to a state whose compactness depends on the overall net charge; at the isoeletric pH (4.7), it is maximally compact, while above and below it is more expanded; and (ii) an exponential phase (in the millisecond time range) leading to the native protein via a transition state(s) possibly involving the formation of a specific salt bridge between Lys10 and Glu70, at the contact between the N and C-terminal helices. Comparison with the widely studied horse cytochrome c allows the discussion of similarities and differences in the folding of two proteins which have the same "fold" despite a very low degree of sequence homology (<30 %).
Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reductase (Pa-NiR). We have measured the (d1--> c) internal eT rate for the wild-type protein and a site-directed mutant (Pa-NiR H327A) which has a substitution in the d1-heme binding pocket; we found the rate of eT to be fast, keT = 2.5 x 10(4) and 3.5 x 10(4) s-1 for the wild-type and mutant Pa-NiR, respectively. We also investigated the photodissociation of CO from the fully reduced proteins and observed microsecond first-order relaxations; these imply that upon breakage of the Fe2+-CO bond, both Pa-NiR and Pa-NiR H327A populate a nonequilibrium state which decays to the ground state with a complex time course that may be described by two exponential processes (k1 = 3 x 10(4) s-1 and k2 = 0.25 x 10(4) s-1). These relaxations do not have a kinetic difference spectrum characteristic of CO recombination, and therefore we conclude that Pa-NiR undergoes structural rearrangements upon dissociation of CO. The bimolecular rate of CO rebinding is 5 times faster in Pa-NiR H327A than in the wild-type enzyme (1.1 x 10(5) M-1 s-1 compared to 2 x 10(4) M-1 s-1), indicating that this mutation in the active site alters the CO diffusion properties of the protein, probably reducing steric hindrance. CO rebinding to the wild-type mixed valence enzyme (c3+d12+) which is very slow (k = 0.25 s-1) is proposed to be rate-limited by the c --> d1 internal eT event, involving the oxidized d1-heme which has a structure characteristic of the fully oxidized and partially oxidized Pa-NiR.
The structures of nitrite reductase from Paracoccus denitrificans GB17 (NiR-Pd) and Pseudomonas aeruginosa (NiR-Pa) have been described for the oxidized and reduced state (Fülöp, V., Moir, J. W. B., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Nurizzo, D., Silvestrini, M. C., Mathieu, M., Cutruzzolà, F., Bourgeois, D., Fülöp, V., Hajdu, J., Brunori, M., Tegoni, M., and Cambillau, C. (1997) Structure 5, 1157-1171; Nurizzo, D., Cutruzzolà, F., Arese, M., Bourgeois, D., Brunori, M., Cambillau, C. , and Tegoni, M. (1998) Biochemistry 37, 13987-13996). Major conformational rearrangements are observed in the extreme states although they are more substantial in NiR-Pd. The four structures differ significantly in the c heme domains. Upon reduction, a His17/Met106 heme-ligand switch is observed in NiR-Pd together with concerted movements of the Tyr in the distal site of the d1 heme (Tyr10 in NiR-Pa, Tyr25 in NiR-Pd) and of a loop of the c heme domain (56-62 in NiR-Pa, 99-116 in NiR-Pd). Whether the reduction of the c heme, which undergoes the major rearrangements, is the trigger of these movements is the question addressed by our study. This conformational reorganization is not observed in the partially reduced species, in which the c heme is partially or largely (15-90%) reduced but the d1 heme is still oxidized. These results suggest that the d1 heme reduction is likely to be responsible of the movements. We speculate about the mechanistic explanation as to why the opening of the d1 heme distal pocket only occurs upon electron transfer to the d1 heme itself, to allow binding of the physiological substrate NO2- exclusively to the reduced metal center.
The structure-function relationships in nitrite reductases, key enzymes in the dissimilatory denitrification pathway which reduce nitrite to nitric oxide (NO), are reviewed in this paper. The mechanisms of NO production are discussed in detail and special attention is paid to new structural information, such as the high resolution structure of the copper- and heme-containing enzymes from different sources. Finally, some implications relevant to regulation of the steady state levels of NO in denitrifiers are presented.
A triple mutant of sperm whale myoglobin (Mb) [Leu(B10) --> Tyr, His(E7) --> Gln, and Thr(E10) --> Arg, called Mb-YQR], investigated by stopped-flow, laser photolysis, crystallography, and molecular dynamics (MD) simulations, proved to be quite unusual. Rebinding of photodissociated NO, O2, and CO from within the protein (in a "geminate" mode) allows us to reach general conclusions about dynamics and cavities in proteins. The 3D structure of oxy Mb-YQR shows that bound O2 makes two H-bonds with Tyr(B10)29 and Gln(E7)64; on deoxygenation, these two residues move toward the space occupied by O2. The bimolecular rate constant for NO binding is the same as for wild-type, but those for CO and O2 binding are reduced 10-fold. While there is no geminate recombination with O2 and CO, geminate rebinding of NO displays an unusually large and very slow component, which is pretty much abolished in the presence of xenon. These results and MD simulations suggest that the ligand migrates in the protein matrix to a major "secondary site," located beneath Tyr(B10)29 and accessible via the motion of Ile(G8)107; this site is different from the "primary site" identified by others who investigated the photolyzed state of wild-type Mb by crystallography. Our hypothesis may rationalize the O2 binding properties of Mb-YQR, and more generally to propose a mechanism of control of ligand binding and dissociation in hemeproteins based on the dynamics of side chains that may (or may not) allow access to and direct temporary sequestration of the dissociated ligand in a docking site within the protein. This interpretation suggests that very fast (picosecond) fluctuations of amino acid side chains may play a crucial role in controlling O2 delivery to tissue at a rate compatible with physiology.
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Nitrite reductase (NiR) from Pseudomonas aeruginosa (EC 1.9.3.2) (NiR-Pa) is a soluble enzyme catalyzing the reduction of nitrite (NO2-) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which each monomer carries one c and one d1 heme. The oxidized and reduced forms of NiR from Paracoccus denitrificans GB17 (previously called Thiosphaera pantotropha) (NiR-Pd) have been described [Fülop, V., et al. (1995) Cell 81, 369-377; Williams, P. A., et al. (1997) Nature 389, 406-412], and we recently reported on the structure of oxidized NiR-Pa at 2.15 A [Nurizzo, D., et al. (1997) Structure 5, 1157-1171]. Although the domains carrying the d1 heme are almost identical in both NiR-Pa and NiR-Pd oxidized and reduced structures, the c heme domains show a different pattern of c heme coordination, depending on the species and the redox state. The sixth d1 heme ligand in oxidized NiR-Pd was found to be Tyr25, whereas in NiR-Pa, the homologuous Tyr10 does not interact directly with Fe3+, but via a hydroxide ion. Furthermore, upon reduction, the axial ligand of the c heme of NiR-Pd changes from His17 to Met108. Finally, in the oxidized NiR-Pa structure, the N-terminal stretch of residues (1-29) of one monomer interacts with the other monomer (domain swapping), which does not occur in NiR-Pd. Here the structure of reduced NiR-Pa is described both in the unbound form and with the physiological product, NO, bound at the d1 heme active site. Although both structures are similar to that of reduced NiR-Pd, significant differences with respect to oxidized NiR-Pd were observed in two regions: (i) a loop in the c heme domain (residues 56-62) is shifted 6 A away and (ii) the hydroxide ion, which is the sixth coordination ligand of the heme, is removed upon reduction and NO binding and the Tyr10 side chain rotates away from the position adopted in the oxidized form. The conformational changes observed in NiR-Pa as the result of reduction are less extensive than those occurring in NiR-Pd. Starting with oxidized structures that differ in many respects, the two enzymes converge, yielding reduced conformations which are very similar to each other, which indicates that the conformational changes involved in catalysis are considerably diverse.
The unfolding of the small cytochrome c551 from the bacterium Pseudomonas aeruginosa has been characterized at equilibrium by circular dichroism (CD) and fluorescence spectroscopy. The process can be described by a two state mechanism and the thermodynamic stability of cytochrome c551 is found to be smaller than that of the larger horse cytochrome c (deltaGw = -8.2 vs. -9.7 kcal/mol); we propose that this finding is related to the absence of an 'omega' loop in the bacterial cytochrome. Cytochrome c551 loses most of its secondary structure at pH 1.5. The acid transition (pKA approximately 2) is highly cooperative (n > or =2); analysis of optical titrations and contact map suggests that (at least) His-16 (proximal Fe3+ ligand) and Glu-70 are both involved in the acid transition. The role of selected hydrophobic, electrostatic and conformational contributions to the overall stability has been investigated by protein engineering. The equilibrium characterization of wild-type and mutant cytochrome c551 supports the view that this small cytochrome is an interesting protein to analyze the thermodynamics and the kinetics of folding in comparison with the widely studied horse cytochrome c.
The equilibrium unfolding pathway of Aplysia apomyoglobin has been studied under various solvent conditions. The protein exhibits a single unfolding transition in acid in contrast to the two transitions observed for the mammalian apomyoglobins with which it shares a common fold but a low level of sequence identity (24%). This acid-unfolded species has considerable residual structure as evidenced by both tryptophan fluorescence and far-UV CD spectroscopy. It remains 40% alpha-helical under low salt conditions (2 mM citrate, 4 degrees C); the folded form is 65% helical. A similar species is observed for the mammalian globins in mild acid conditions. Titration with GdnHCl at pH 7 reveals two unfolding transitions, the first having common features with that observed in acid and the second resulting in a completely unfolded state. Under the same conditions, urea unfolds the protein completely in an apparently single cooperative transition. Assuming a simple three-state model (F<-->I<-->U), data from GdnHCl and urea titrations over a range of pH conditions were used to derive values for the apparent stability (delta Gw(app) and solvent accessibility (n(app)) of the folded (F) and intermediate (I) forms of the protein. Urea titrations were then repeated over a range of KCl concentrations in order to understand the contribution of Cl- to the different unfolding activity of GdnHCl. A three-state scheme is justified when changes in delta G(w(app)) occur without changes in n(app). The change in free energy of folding of I<-->F (delta Gw(F/I)) decreases to 0 at pH 4 as expected from the acid unfolding curve. delta Gw(I/U) reaches its maximum at pH 4.5, the isoelectric point of the protein. Variations of this value with pH and chloride are as much as 3 kcal mol-1 and correlate closely with changes in n(app) although there is no change in the alpha-helical content of I across the pH range. This observation is interpreted here as a deviation of the unfolding of the I state of Aplysia apomyoglobin from a cooperative behaviour.
BACKGROUND: Nitrite reductase from Pseudomonas aeruginosa (NiR-Pa) is a dimer consisting of two identical 60 kDa subunits, each of which contains one c and one d1 heme group. This enzyme, a soluble component of the electron-transfer chain that uses nitrate as a source of energy, can be induced by the addition of nitrate to the bacterial growth medium. NiR-Pa catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO); in vitro, both cytochrome c551 and azurin are efficient electron donors in this reaction. NiR is a key denitrification enzyme, which controls the rate of the production of toxic nitric oxide (NO) and ultimately regulates the release of NO into the atmosphere. RESULTS: The structure of the orthorhombic form (P2(1)2(1)2) of oxidized NiR-Pa was solved at 2.15 A resolution, using molecular replacement with the coordinates of the NiR from Thiosphaera pantotropha (NiR-Tp) as the starting model. Although the d1-heme domains are almost identical in both enzyme structures, the c domain of NiR-Pa is more like the classical class I cytochrome-c fold because it has His51 and Met88 as heme ligands, instead of His17 and His69 present in NiR-Tp. In addition, the methionine-bearing loop, which was displaced by His17 of the NiR-Tp N-terminal segment, is back to normal in our structure. The N-terminal residues (5/6-30) of NiR-Pa and NiR-Tp have little sequence identity. In Nir-Pa, this N-terminal segment of one monomer crosses the dimer interface and wraps itself around the other monomer. Tyr10 of this segment is hydrogen bonded to an hydroxide ion--the sixth ligand of the d1-heme Fe, whereas the equivalent residue in NiR-Tp, Tyr25, is directly bound to the Fe. CONCLUSIONS: Two ligands of hemes c and d1 differ between the two known NiR structures, which accounts for the fact that they have quite different spectroscopic and kinetic features. The unexpected domain-crossing by the N-terminal segment of NiR-Pa is comparable to that of 'domain swapping' or 'arm exchange' previously observed in other systems and may explain the observed cooperativity between monomers of dimeric NiR-Pa. In spite of having similar sequence and fold, the different kinetic behaviour and the spectral features of NiR-Pa and NiR-Tp are tuned by the N-terminal stretch of residues. A further example of this may come from another NiR, from Pseudomonas stutzeri, which has an N terminus very different from that of the two above mentioned NiRs.
In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d1 heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd1 NiR in which Tyr10 has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fulop, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369-377) that the topologically homologous Tyr25 plays a crucial role in controlling the activity of the cd1 NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr10 with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe3+ of the d1 heme is provided by different side-chains in different species.