Tyrosine 92 of aristolochene synthase directs cyclisation of farnesyl pyrophosphate

A mutant of Aristolochene Synthase (AS), in which Tyr 92 was replaced by Val, produced the alicyclic beta-(E)-farnesene as the major product, indicating that cyclisation of FPP is controlled by Tyr 92 in AS.     

Electron magnetic resonance and density functional theory study of room temperature X-irradiated β-D-fructose single crystals

Stable free radical formation in fructose single crystals X-irradiated at room temperature was investigated using Q-band electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and ENDOR induced EPR (EIE) techniques. ENDOR angular variations in the three main crystallographic planes allowed an unambiguous determination of 12 proton HFC tensors. From the EIE studies, these hyperfine interactions were assigned to six different radical species, labeled F1-F6. Two of the radicals (F1 and F2) were studied previously by Vanhaelewyn et al. [Vanhaelewyn, G. C. A. M.; Pauwels, E.; Callens, F. J.; Waroquier, M.; Sagstuen, E.; Matthys, P. J. Phys. Chem. A 2006, 110, 2147.] and Tarpan et al. [Tarpan, M. A.; Vrielinck, H.; De Cooman, H.; Callens, F. J. J. Phys. Chem. A 2009, 113, 7994.]. The other four radicals are reported here for the first time and periodic density functional theory (DFT) calculations were used to aid their structural identification. For the radical F3 a C3 carbon centered radical with a carbonyl group at the C4 position is proposed. The close similarity in HFC tensors suggests that F4 and F5 originate from the same type of radical stabilized in two slightly different conformations. For these radicals a C2 carbon centered radical model with a carbonyl group situated at the C3 position is proposed. A rather exotic C2 centered radical model is proposed for F6.     

Determinants of cyanuric acid and melamine assembly in water

While the recognition of cyanuric acid (CA) by melamine (M) and their derivatives has been known to occur in both water and organic solvents for some time, analysis of CA/M assembly in water has not been reported (Ranganathan, A.; Pedireddi, V. R.; Rao, C. N. R. J. Am. Chem. Soc.1999, 121, 1752-1753; Mathias, J. P.; Simanek, E. E.; Seto, C. T.; Whitesides, G. M. Macromol. Symp.1994, 77, 157-166; Zerkowski, J. A.; MacDonald, J. C.; Seto, C. T.; Wierda, D. A.; Whitesides, G. M. J. Am. Chem. Soc.1994, 116, 2382-2391; Mathias, J. P.; Seto, C. T.; Whitesides, G. M. Polym. Prepr.1993, 34, 92-93; Seto, C. T.; Whitesides, G. M. J. Am. Chem. Soc.1993, 115, 905-916; Zerkowski, J. A.; Seto, C. T.; Whitesides, G. M. J. Am. Chem. Soc.1992, 114, 5473-5475; Seto, C. T.; Whitesides, G. M. J. Am. Chem. Soc.1990, 112, 6409-6411; Wang, Y.; Wei, B.; Wang, Q. J. Chem. Cryst.1990, 20, 79-84; ten Cate, M. G. J.; Huskens, J.; Crego-Calama, M.; Reinhoudt, D. N. Chem.-Eur. J.2004, 10, 3632-3639). We have examined assembly of CA/M, as well as assembly of soluble trivalent CA and M derivatives (TCA/TM), in aqueous solvent, using a combination of solution phase NMR, isothermal titration and differential scanning calorimetry (ITC/DSC), cryo-transmission electron microscopy (cryo-TEM), and synthetic chemistry. While the parent heterocycles coprecipitate in water, the trivalent system displays more controlled and cooperative assembly that occurs at lower concentrations than the parent and yields a stable nanoparticle suspension. The assembly of both parent and trivalent systems is rigorously 1:1 and proceeds as an exothermic, proton-transfer coupled process in neutral pH water. Though CA and M are considered canonical hydrogen-bonding motifs in organic solvents, we find that their assembly in water is driven in large part by enthalpically favorable surface-area burial, similar to what is observed with nucleic acid recognition. There are currently few synthetic systems capable of robust molecular recognition in water that do not rely on native recognition motifs, possibly due to an incomplete understanding of recognition processes in water. This study establishes a detailed conceptual framework for considering CA/M heterocycle recognition in water which enables the future design of molecular recognition systems that function in water.     

Stabilisation of transition states prior to and following eudesmane cation in aristolochene synthase

The mechanistic details of the cyclisation of farnesylpyrophosphate (FPP) by aristolochene synthase (AS) from Penicillium roqueforti have only recently begun to emerge, mainly through the analysis of the reaction products generated by AS-mutants. The reaction proceeds through several intermediates including germacrene A and eudesmane cation. Previous work suggested that the side chain of phenylalanine 178 promoted the conversion of eudesmane cation to aristolochene. We now report that the catalytic function of this residue during the conversion of eudesmane cation to aristolochene is mainly due to the large size of its side chain, which facilitates the hydride shift from C2 to C3, rather than its aromatic character. In addition, F178 appears to control the regioselectivity of the final deprotonation step and, together with F112, helps stabilise the developing positive charge on C1 after the expulsion of pyrophosphate from the substrate. These results complete a screen of likely active-site aromatic residues and establish their respective roles in the conversion of FPP to aristolochene.     

Evaluation of Solvent Accessibility to the [Fe(4)S(4)] Binding Pocket in Native and Tyr19 Mutant High Potential Iron Proteins by (1)H-(15)N HMQC and (19)F NMR Experiments

The solvent accessibility of Chromatium vinosumhigh potential iron protein (HiPIP) has been investigated by use of (1)H-(15)N HMQC, and (19)F NMR spectroscopy. These NMR experiments indicate that solvent accessibility to the cluster core is similar, and minimal, for the reduced and oxidized states of native HiPIP, but increases significantly for mutant proteins (Tyr19Leu and Tyr19His). These results support a proposed role [Agarwal, A.; Li, D.; Cowan, J. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9440-9444] for Tyr19 in maintaining hydrolytic stability of the [Fe(4)S(4)] cluster, and demonstrate a general strategy for mapping out the solvent accessibility of protein-bound metalloredox prosthetic centers.     

Dual role for phenylalanine 178 during catalysis by aristolochene synthase

A mutant of aristolochene synthase, in which Phe 178 was replaced by Val, produced significant amounts of alpha-and beta-farnesene as well as alpha and beta-selinene and selina-4,11-diene, suggesting that Phe 178 is involved in the stabilisation of transition states preceding germacrene A and following eudesmane cation.     

Product distribution of peroxynitrite decay as a function of pH, temperature, and concentration.

The decay of peroxynitrite [O=NOO(-), oxoperoxonitrate(1-)] was examined as a function of concentration (0.050-2.5 mM), temperature (5-45 degrees C), and pH (2.2-10.0). Below 5 degrees C and pH 7, little amounts of the decomposition products nitrite and dioxygen are formed, even when the peroxynitrite concentration is high (2.5 mM). Instead, approximately > or =90% isomerizes to nitrate. At higher pH, decomposition increases at the expense of isomerization, up to nearly 80% at pH 10.0 at 5 degrees C and 90% at 45 degrees C. Much less nitrite and dioxygen per peroxynitrite are formed when the peroxynitrite concentration is lower; at 50 microM and pH 10.2, < or =40% decomposes. In contrast to two other reports (Pfeiffer, S.; Gorren, A. C. F.; Schmidt, K.; Werner, E. R.; Hansert, B.; Bohle, D. S.; Mayer, B. J. Biol. Chem. 1997, 272, 3465-3470, and Coddington, J. W.; Hurst, J. K.; Lymar, S. V. J. Am. Chem. Soc. 1999, 121, 2438-2443), we find that the extent of decomposition is dependent on the peroxynitrite concentration.     

Origin of the nonplanarity of tetrafluoro cyclobutadiene, C4F4

Density functional theory as well as M?ller-Plesset investigations has been carried out on tetrafluoro cyclobutadiene, C4F4, to explore the origin of its nonplanarity. Although Petersson et al. (Petersson, E. J.; Fanuele, J. C.; Nimlos, M. R.; Lemal, D. M.; Ellison, G. B.; Radziszewski, J. G. J. Am. Chem. Soc. 1997, 119, 11122-11123) had earlier predicted a nonplanar geometry of this compound on the basis of spectral and bond orbital analysis, the explanation of the same from a more fundamental point of view is still missing. In the present study, we provide a heuristic explanation for the origin of nonplanarity of C4F4. The two major driving forces behind this nonplanar geometry are the unusual aromaticity of this cyclic homoatomic 4pi electron system and the second-order Jahn-Teller effect (SOJTE). These driving forces can well be explained by various energy and density parameters and also by nucleus-independent chemical shift (NICS) values. Aromaticity of a cyclic homoatomic 4pi electron system is quite remarkable. The enhancement of pi- delocalization as evidenced from molecular orbital analysis may be attributed to s-ppi mixing in nonplanar C4F4.     

Rate constants and H atom branching ratios of the gas-phase reactions of methylidyne CH(X2Pi) radical with a series of alkanes

The reactions of the CH radical with several alkanes were studied, at room temperature, in a low-pressure fast-flow reactor. CH(X2Pi, v = 0) radicals were obtained from the reaction of CHBr(3) with potassium atoms. The overall rate constants at 300 K are (0.76 +/- 0.20) x 10(-10) [Fleurat-Lessard, P.; Rayez, J. C.; Bergeat, A.; Loison, J. C. Chem. Phys. 2002, 279, 87],1 (1.60 +/- 0.60) x 10(-10)[Galland, N.; Caralp, F.; Hannachi, Y.; Bergeat, A.; Loison, J.-C. J. Phys. Chem. A 2003, 107, 5419],2 (2.20 +/- 0.80) x 10(-10), (2.80 +/- 0.80) x 10(-10), (3.20 +/- 0.80) x 10(-10), (3.30 +/- 0.60) x 10(-10), and (3.60 +/- 0.80) x 10(-10) cm3 molecule(-1) s(-1), (errors refer to +/-2sigma) for methane, ethane, propane, n-butane, n-pentane, neo-pentane, and n-hexane respectively. The experimental overall rate constants correspond to those obtained using a simple classical capture theory. Absolute atomic hydrogen production was determined by V.U.V. resonance fluorescence, with H production from the CH + CH4 reaction being used as a reference. Observed H branching ratios were for CH4, 1.00[Fleurat-Lessard, P.; Rayez, J. C.; Bergeat, A.; Loison, J. C. Chem. Phys. 2002, 279, 87];1 C(2)H(6), 0.22 +/- 0.08 [Galland, N.; Caralp, F.; Hannachi, Y.; Bergeat, A.; Loison, J.-C. J. Phys. Chem. A 2003, 107, 5419];2 C(3)H(8), 0.19 +/- 0.07; C(4)H(10) (n-butane), 0.14 +/- 0.06; C(5)H(12) (n-pentane), 0.52 +/- 0.08; C(5)H(12) (neo-pentane), 0.51 +/- 0.08; C(5)H(12) (iso-pentane), 0.12 +/- 0.06; C(6)H(14) (n-hexane), 0.06 +/- 0.04.     

Synthesis and properties of a fluorene-capped isotruxene: a new unsymmetrical star-shaped pi-system

An unsymmetrical star-shaped pi-system (1) with an isotruxene core and three fluorene arms has been synthesized, and its photophysical, electrochemical, and thermochemical properties have been investigated and compared with the corresponding symmetrical truxene derivatives 2a-d (Kanibolotsky, A. L.; Berridge, R.; Skabara, P. J.; Perepichka, I. F.; Bradley, D. D. C.; Koeberg, M. J. Am. Chem. Soc. 2004, 126, 13695-13702). Stronger electronic couplings between the arms in 1 vs 2a-d lead to lower optical band gap, larger splitting in oxidation potentials, and superior thermostability. [structure: see text]     

Evaluation of data for atmospheric models: master equation/RRKM calculations on the combination reaction, BrO + NO2 --> BrONO2, a conundrum

Experimental data for the title reaction were modeled using master equation (ME)/RRKM methods based on the Multiwell suite of programs. The starting point for the exercise was the empirical fitting provided by the NASA (Sander, S. P.; Finlayson-Pitts, B. J.; Friedl, R. R.; Golden, D. M.; Huie, R. E.; Kolb, C. E.; Kurylo, M. J.; Molina, M. J.; Moortgat, G. K.; Orkin, V. L.; Ravishankara, A. R. Chemical Kinetics and Photochemical Data for Use in Atmospheric Studies, Evaluation Number 15; Jet Propulsion Laboratory: Pasadena, California, 2006)1 and IUPAC (Atkinson, R.; Baulch, D. L.; Cox, R. A.; R. F. Hampson, J.; Kerr, J. A.; Rossi, M. J.; Troe, J. J. Phys. Chem. Ref. Data 2000, 29, 167)2 data evaluation panels, which represents the data in the experimental pressure ranges rather well. Despite the availability of quite reliable parameters for these calculations (molecular vibrational frequencies (Parthiban, S.; Lee, T. J. J. Chem. Phys. 2000, 113, 145)3 and a value (Orlando, J. J.; Tyndall, G. S. J. Phys. Chem. 1996, 100, 19398)4 of the bond dissociation energy, D298(BrO-NO2) = 118 kJ mol-1, corresponding to DeltaH0o = 114.3 kJ mol-1 at 0 K) and the use of RRKM/ME methods, fitting calculations to the reported data or the empirical equations was anything but straightforward. Using these molecular parameters resulted in a discrepancy between the calculations and the database of rate constants of a factor of ca. 4 at, or close to, the low-pressure limit. Agreement between calculation and experiment could be achieved in two ways, either by increasing DeltaH0o to an unrealistically high value (149.3 kJ mol-1) or by increasing DeltaEd, the average energy transferred in a downward collision, to an unusually large value (>5000 cm-1). The discrepancy could also be reduced by making all overall rotations fully active. The system was relatively insensitive to changing the moments of inertia in the transition state to increase the centrifugal effect. The possibility of involvement of BrOONO was tested and cannot account for the difficulties of fitting the data.     

Shock tube investigation of CH3 + CH3OCH3

The title reaction has been investigated in a diaphragmless shock tube by laser schlieren densitometry over the temperature range 1163-1629 K and pressures of 60, 120, and 240 Torr. Methyl radicals were produced by dissociation of 2,3-butanedione in the presence of an excess of dimethyl ether. Rate coefficients for CH(3) + CH(3)OCH(3) were obtained from simulations of the experimental data yielding the following expression which is valid over the range 1100-1700 K: k = (10.19 ± 3.0)T(3.78)?exp((-4878/T)) cm(3) mol(-1)s(-1). The experimental results are in good agreement with estimates by Curran and co-workers [Fischer, S. L.; Dryer, F. L.; Curran, H. J. Int. J. Chem. Kinet.2000, 32 (12), 713-740. Curran, H. J.; Fischer, S. L.; Dryer, F. L. Int. J. Chem. Kinet.2000, 32 (12), 741-759] but about a factor of 2.6 lower than those of Zhao et al. [Zhao, Z.; Chaos, M.; Kazakov, A.; Dryer, F. L. Int. J. Chem. Kinet.2008, 40 (1), 1-18].     

Validation studies of the site-directed docking program LibDock

The performance of the site-features docking algorithm LibDock has been evaluated across eight GlaxoSmithKline targets as a follow-up to a broad validation study of docking and scoring software (Warren, G. L.; Andrews, W. C.; Capelli, A.; Clarke, B.; Lalonde, J.; Lambert, M. H.; Lindvall, M.; Nevins, N.; Semus, S. F.; Senger, S.; Tedesco, G.; Walls, I. D.; Woolven, J. M.; Peishoff, C. E.; Head, M. S. J. Med. Chem. 2006, 49, 5912-5931). Docking experiments were performed to assess both the accuracy in reproducing the binding mode of the ligand and the retrieval of active compounds in a virtual screening protocol using both the DJD (Diller, D. J.; Merz, K. M., Jr. Proteins 2001, 43, 113-124) and LigScore2 (Krammer, A. K.; Kirchoff, P. D.; Jiang, X.; Venkatachalam, C. M.; Waldman, M. J. Mol. Graphics Modell. 2005, 23, 395-407) scoring functions. This study was conducted using DJD scoring, and poses were rescored using all available scoring functions in the Accelrys LigandFit module, including LigScore2. For six out of eight targets at least 30% of the ligands were docked within a root-mean-square difference (RMSD) of 2.0 A for the crystallographic poses when the LigScore2 scoring function was used. LibDock retrieved at least 20% of active compounds in the top 10% of screened ligands for four of the eight targets in the virtual screening protocol. In both studies the LigScore2 scoring function enhanced the retrieval of crystallographic poses or active compounds in comparison with the results obtained using the DJD scoring function. The results for LibDock accuracy and ligand retrieval in virtual screening are compared to 10 other docking and scoring programs. These studies demonstrate the utility of the LigScore2 scoring function and that LibDock as a feature directed docking method performs as well as docking programs that use genetic/growing and Monte Carlo driven algorithms.     

Dynamic hydrogen-bonding network in the distal pocket of the nitrosyl complex of Pseudomonas aeruginosa cd1 nitrite reductase

cd(1) nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d(1)-heme cofactor, found only in this class of enzymes, where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d(1)-heme to avoid enzyme inhibition by the formation of ferrous-nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d(1)-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761-14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d(1)-heme NO complex from Pseudomonas aeruginosa cd(1) NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the "distal" side of the heme (Tyr(10), His(327), and His(369)), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron-nuclear double resonance (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d(1)-heme complex of cd(1) NIR forms H-bonds with Tyr(10) and His(369), whereas the second conserved histidine, His(327), appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr(10) is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His(369) is closer to the NO, whereas mutation of both distal histidines displaces Tyr(10), removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed.     

Marked difference in the electronic structure of cyanide-ligated ferric protoglobins and myoglobin due to heme ruffling

Electron paramagnetic resonance experiments reveal a significant difference between the principal g values (and hence ligand-field parameters) of the ferric cyanide-ligated form of different variants of the protoglobin of Methanosarcina acetivorans (MaPgb) and of horse heart myoglobin (hhMb). The largest principal g value of the ferric cyanide-ligated MaPgb variants is found to be significantly lower than for any of the other globins reported so far. This is at least partially caused by the strong heme distortions as proven by the determination of the hyperfine interaction of the heme nitrogens and mesoprotons. Furthermore, the experiments confirm recent theoretical predictions [Forti, F.; Boechi, L., Bikiel, D., Martí, M.A.; Nardini, M.; Bolognesi, M.; Viappiani, C.; Estrin, D.; Luque, F. J. J. Phys. Chem. B2011, 115, 13771-13780] that Phe(G8)145 plays a crucial role in the ligand modulation in MaPgb. Finally, the influence of the N-terminal 20 amino-acid chain on the heme pocket in these protoglobins is also proven.     

Theoretical evidence for C-F bond activation by a fluoro-calix[4]pyrrole-tert-amine macrocycle

Density functional theory as well as highly correlated ab initio molecular orbital theory was used to explore the possibility of activating C-F bonds in fluoroalkanes by organic macrocycles. The results indicate that the reaction between fluoro-calix [4]pyrrole-tert-amine and CH3F via a Menshutkin displacement mechanism is highly favorable and competitive from a thermochemical point of view with the very efficient C-Cl activation by a simple macrocyclic amine recently reported in the literature (Stanger, L. J.; Noll, B. C.; Gonzalez, C.; Marquez, M.; Smith, B. D. J. Am. Chem. Soc. 2005, 127, 4184).     

Reaction of ethylene with hydroxyl radicals: a theoretical study

Ab initio calculations of portions of the C2H5O potential energy surface critical to the title reaction are presented. These calculations are based on QCISD geometries and frequencies and RQCISD(T) energies extrapolated to the complete-basis-set limit. Rate coefficients for the reaction of C2H4 with OH are calculated using this surface and the two transition-state model of Greenwald and co-workers [J. Phys. Chem. A 2005, 109, 6031] for the association of OH with C2H4. The present calculations reproduce most of the experimental data, including the temperature and pressure dependence of the rate coefficients, with only a small (0.4 kcal/mol) adjustment to the energy barrier for direct hydrogen abstraction. We confirm the importance of this channel above 800 K and find that a significant fraction of the total rate coefficient (approximately 10%) is due to the formation of vinyl alcohol above this temperature. Calculations of the vinyl alcohol channel are consistent with the recent observation of this molecule in low-pressure flames [Taatjes, C. A.; Hansen, N.; McIlroy, A.; Miller, J. A.; Senosiain, J. P.; Klippenstein, S. J.; Qi, F.; Sheng, L.; Zhang, Y.; Cool, T. A.; Wang, J.; Westmoreland, P. R.; Law, M. E.; Kasper, T.; Kohse-H?inghaus, K. Science 2005, 308, 1887] and suggest that this reaction should be included in hydrocarbon oxidation mechanisms.     

Bound state potential energy surface construction: ab initio zero-point energies and vibrationally averaged rotational constants

Collins' method of interpolating a potential energy surface (PES) from quantum chemical calculations for reactive systems (Jordan, M. J. T.; Thompson, K. C.; Collins, M. A. J. Chem. Phys. 1995, 102, 5647. Thompson, K. C.; Jordan, M. J. T.; Collins, M. A. J. Chem. Phys. 1998, 108, 8302. Bettens, R. P. A.; Collins, M. A. J. Chem. Phys. 1999, 111, 816) has been applied to a bound state problem. The interpolation method has been combined for the first time with quantum diffusion Monte Carlo calculations to obtain an accurate ground state zero-point energy, the vibrationally average rotational constants, and the vibrationally averaged internal coordinates. In particular, the system studied was fluoromethane using a composite method approximating the QCISD(T)/6-311++G(2df,2p) level of theory. The approach adopted in this work (a) is fully automated, (b) is fully ab initio, (c) includes all nine nuclear degrees of freedom, (d) requires no assumption of the functional form of the PES, (e) possesses the full symmetry of the system, (f) does not involve fitting any parameters of any kind, and (g) is generally applicable to any system amenable to quantum chemical calculations and Collins' interpolation method. The calculated zero-point energy agrees to within 0.2% of its current best estimate. A0 and B0 are within 0.9 and 0.3%, respectively, of experiment.     

Extension of the renormalized coupled-cluster methods exploiting left eigenstates of the similarity-transformed hamiltonian to open-shell systems: a benchmark study

The recently formulated completely renormalized coupled-cluster method with singles, doubles, and noniterative triples, exploiting the biorthogonal form of the method of moments of coupled-cluster equations (Piecuch, P.; W?och, M. J. Chem. Phys. 2005, 123, 224105; Piecuch, P.; W?och, M.; Gour, J. R.; Kinal, A. Chem. Phys. Lett. 2006, 418, 467), termed CR-CC(2,3), is extended to open-shell systems. Test calculations for bond breaking in the OH radical and the F2+ ion and singlet-triplet gaps in the CH2, HHeH, and (HFH)- biradical systems indicate that the CR-CC(2,3) approach employing the restricted open-shell Hartree--Fock (ROHF) reference is significantly more accurate than the widely used CCSD(T) method and other noniterative triples coupled-cluster approximations without making the calculations substantially more expensive. A few molecular examples, including the activation energies of the C2H4 + H --> C2H5 forward and reverse reactions and the triplet states of the CH2 and H2Si2O2 biradicals, are used to show that the dependence of the ROHF-based CR-CC(2,3) energies on the method of canonicalization of the ROHF orbitals is, for all practical purposes, negligible.