Characterization of Recombinant Horseradish Peroxidase C and three Site-Directed mutants, F41V, F41W, and R38K by Resonance Raman Spectroscopy

Smulevich, Giulietta; Paoli, M.; Burke, Julian F.; Sanders, Stephen A.; Thorneley, R. N. F.; Smith, Andrew T.

doi:10.1021/bi00189a046

Cited by 100 publications

(123 citation statements)

References 54 publications

(69 reference statements)

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“…The m(C=C) vinyl stretching modes are enhanced via the A-term (Franck-Condon mechanism); however, these bands are also observed in Q band excitation. This is consistent with previous observations for various heme containing peroxidases-namely CcP [24], HRPC [25], and Coprinus cinereus peroxidase (CIP) [26]-despite the fact that vinyl stretching modes are not expected to be enhanced a priori with visible excitation. The complete assignment of the high-frequency region is reported in Table S1 of the ESM.…”

Section: Spectroscopy At Room Temperaturesupporting

confidence: 92%

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

et al. 2010

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The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 a-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.

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Section: Spectroscopy At Room Temperaturesupporting

confidence: 92%

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

et al. 2010

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“…The 3 band at 1503 cm Ϫ1 together with the 2 mode at 1574 cm Ϫ1 (only the mean frequency can be given, due to overlap with the 2 modes of other heme species), 19 at 1576 cm Ϫ1 , and 10 at 1638 cm Ϫ1 are consistent with the QS heme state being the dominant heme form in Ca 2ϩ -depleted HRPC. The low frequency of the 11 mode at 1548 cm Ϫ1 compared with the high frequencies observed for the other modes is a characteristic feature of QS states that had been noted previously (22)(23)(24) and is a further demonstration that the QS state is the major heme species.…”

Section: Roles Of the Calcium Ions In Hrpcmentioning

confidence: 60%

“…3B. The spectrum of the native has been reported previously and found to be a mixture of 5-coordinate (5-c) and 6-c heme forms, both of which display anomalously high core size marker band frequencies (19). On the basis of this observation and other data available for HRPC, it has been proposed that resting HRPC at room temperature cannot be adequately described by pure HS heme states and that the two heme forms should be considered as QS states having a very low level of IS state admixed with the HS state (20,21).…”

Section: Electronic Absorption and Resonance Raman Of Ferric Enzymes-mentioning

confidence: 60%

“…The predominant QS species co-exists with a 5-c HS ( 3 at 1493 cm Ϫ1 , 11 at 1553 cm Ϫ1 , and 10 at 1631 cm Ϫ1 ) and a 6-c HS form ( 3 at 1485 cm Ϫ1 and 10 at 1622 cm Ϫ1 ). Moreover, excitation at 568.2 nm, in resonance with the ␣-band of a 6-c LS heme state, gives rise to spectral changes consistent with the presence of a LS species ( 11 at 1560 cm Ϫ1 , 19 at 1586 cm Ϫ1 , and 10 at 1640 cm Ϫ1 ) with band frequencies higher than those of the QS state. However, the LS species is present in such a small amount that it cannot be detected in the electronic absorption spectrum.…”

Section: Roles Of the Calcium Ions In Hrpcmentioning

confidence: 87%

“…the two hexacoordinate states, 6-c HS and 6-c QS, which characterize the native BHA complex (19,34). In fact, subtraction of the spectrum of the native complex from that of the Ca 2ϩ -depleted form (data not shown) indicates an increased proportion of the 6-c HS state ( 3 at 1486 cm Ϫ1 , 2 at 1562 cm Ϫ1 , 37 at 1579 cm Ϫ1 , and 10 at 1615 cm Ϫ1 ) in the Ca 2ϩ -depleted complex.…”

Section: Carbon Monoxide and Bha Binding To Camentioning

confidence: 99%

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The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

Howes

Feis

Raimondi³

et al. 2001

Journal of Biological Chemistry

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The extent to which the structural Ca 2؉ ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca 2؉ ion. The Fe(III) heme states are modified upon Ca 2؉ depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca 2؉ -depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca 2؉ depletion are easily reversed. Contrary to the native protein, the Ca 2؉ -depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the (Fe-Im) stretching mode downshifts 27 cm ؊1 upon Ca 2؉ depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca 2؉ -depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca 2؉ depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca 2؉ ion as that which is lost upon Ca 2؉ depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.Peroxidases of the plant peroxidase superfamily are hemecontaining enzymes that oxidize a variety of aromatic molecules in the presence of hydrogen peroxide. They include peroxidases of plant, fungal, and prokaryotic origins that can be divided into three classes based on sequence alignment (1).Additional support for such a classification was gained as the crystal structures of representative enzymes of each class became available. Class I contains bacterial peroxidases and peroxidases from plant mitochondria and chloroplasts, for example most ascorbate peroxidases and cytochrome c peroxidase. Class II contains extracellular fungal peroxidases such as Coprinus cinereus peroxidase (a peroxidase essentially identical to Arthromyces ramosus peroxidase) and lignin-degrading peroxidases. Class III contains secretory plant peroxidases, typified by the classical horseradish peroxidase isoenzyme C (HRPC). The peroxidases of classes II and III share a number of structural elements considered to be of importance for maintaining protein stability and activity. These include calciumbinding sites proximal and distal to the heme and four disulfide bridges (1-5); the latter was in different locations in class II with respect to class III peroxidases. These features are absent in class I peroxidases. Class III peroxidases also have a loop insertion in the sequence, composed of the DЈ, FЈ, and FЉ helices, not found in the other classes. The relationship between calcium binding and enzyme inactivation has been treated primarily by studies on lignin peroxidase (LIP) (6, 7), manganese peroxidase (MNP) (8, 9), and HRPC (10, 11). Thermal (6) and alkaline (7) i...

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Refinement of 3D models of horseradish peroxidase isoenzyme C: Predictions of 2D NMR assignments and substrate binding sites

et al. 1996

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In this study, two alternative three-dimensional (3D) models of horseradish peroxidase (HRP-C)-differing mainly in the structure of a long untemplated insertion-were refined, systematically assessed, and used to make predictions that can both guide and be tested by future experimental studies. A key first step in the model-building process was a procedure for multiple sequence alignment based on structurally conserved regions and key conserved residues, including those side chains providing ligands to the two Ca2+ binding sites. The model refinements reported here include (1) optimization of side-chain conformations; (3) addition of structural waters using a template-independent procedure; (2) structural refinement of the untemplated 34 amino acid insertion located between the F and G helices, using both energy criteria and NMR data; (4) unconstrained energy optimization of the refined models. Using these procedures, two refined structures of HRP-C were obtained, differing mainly in the conformation of this long insertion. The presence of residues in this insertion that could potentially interact with bound substrates suggests a functional role that may be related to the general ability of class III peroxidases to form stable 1:1 complexes with a variety of substrates. The structural validity of the models was systematically assessed by a variety of criteria. Most notably, the ProsaII z scores and Profiles 3D scores of the two HRP-C models indicated that they are significantly better than would be obtained by simple amino acid replacement, using any of the known structures as a template. These two 3D HRP-C models, were then used to predict candidate residues for the assignment of NOESY cross-peaks previously noted in 2D-NMR studies. Specifically, the residues known as Ile X, Phe A, Phe B, aliphatic residue Q, and Ile T. Candidate substrate binding sites were also identified and compared with experimentally based predictions. This work is timely because new X-ray structures are anticipated that will facilitate the validation of these procedures.

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Characterization of Recombinant Horseradish Peroxidase C and three Site-Directed mutants, F41V, F41W, and R38K by Resonance Raman Spectroscopy

Cited by 100 publications

References 54 publications

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125

The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

Refinement of 3D models of horseradish peroxidase isoenzyme C: Predictions of 2D NMR assignments and substrate binding sites

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