Nature of the iron-ligand bond in ferrous low spin hemoproteins studied by resonance Raman scattering

Kitagawa, Teizo; Kyōgoku, Yoshimasa; Iizuka, Tetsutarō; Mi, Saito

doi:10.1021/ja00433a019

Cited by 163 publications

(107 citation statements)

References 7 publications

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“…In wild-type P450 cam , n 2 and n 3 appeared at 1562 cm 21 and 1467 cm 21 , respectively, reflecting the five-coordinate and high-spin iron [47,48]. The n 2 and n 3 lines for C357H were shifted to higher frequencies (1589 and 1491 cm 21 , respectively), which are typical of the ferrous low-spin heme [49]. We note, however, that a minor line is observed at 1470 cm 21 for C357H.…”

Section: Coordination Structure Of Ferrous C357hmentioning

confidence: 96%

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam

Shiro¹,

Takahashi²,

Hori³

et al. 2001

Eur J Biochem

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To investigate the functional and structural roles of the proximal thiolate ligand in cytochrome P450 cam , we prepared the C357H mutant of the enzyme in which the axial cysteine residue (Cys357) was replaced with a histidine residue. We obtained the unstable C357H mutant by developing a new preparation procedure involving in vitro folding of P450 cam from the inclusion bodies. The C357H mutant in the ferrous-CO form exhibited the Soret peak at 420 nm and the Fe-CO stretching line at 498 cm 21 , indicating a neutral histidine residue as the axial ligand. However, another internal ligand is coordinated to the heme iron as the sixth ligand in the ferric and ferrous forms of the C357H mutant, suggesting the collapse of the substratebinding site. The C357H mutant showed no catalytic activity for camphor hydroxylation and the reduced heterolytic/ homolytic ratio of the O±O bond scission in the reaction with cumene hydroperoxide. The present observations indicate that the thiolate coordination in P450 cam is important for the construction of the heme pocket and the heterolysis of the O±O bond.Keywords: P450; CPO; P420; oxygen activation; axial push effect.Heme-thiolate enzymes such as cytochrome P450 (P450) and chloroperoxidase from Caldariomyces fumago (CPO) activate heme-bound dioxygen and hydrogen peroxide and insert oxygen atoms into various hydrocarbons [1±4]. The enzymes have attracted much interest because the heterolytic O±O bond scission of the heme-bound dioxygen and the C±H bond activation of inactivated hydrocarbons are difficult in mild conditions [1,5]. As the enzymes have an anionic thiolate as the axial heme ligand [6,7], the strong electron donation of the thiolate is supposed to facilitate the heterolysis of the dioxygen that coordinates trans to the thiolate ligand [8,9]. The importance of the thiolate coordination for the activities of these enzymes is exemplified by their inactivated forms (P420 and C420) which lack the thiolate coordination [10,11]. However, the roles of Fe±S bond that are important to understand the molecular mechanism of the enzyme activities are still controversial [12,13].To clarify the roles of the thiolate coordination in the heme-thiolate enzymes, we replaced the axial histidine of myoglobin (Mb) with cysteine (H93C), and investigated its catalytic reactivities [14,15]. We observed that the thiolate ligand of Mb H93C accelerated the heterolysis of the O±O bond of cumene hydroperoxide and enhanced the hydrogen peroxide supported epoxidation of styrene [15]. These observations allowed us to propose that the thiolate ligation in heme proteins encourages the heterolytic cleavage of the O±O bond [15]. Our proposal is further confirmed by Higuchi et al. who demonstrated that the ligation of alkyl thiolate to iron porphyrin facilitates the O±O bond heterolysis of alkyl peracid in hydrophobic solvent [16,17]. These model works indicate the importance of the thiolate coordination in the heterolytic O±O bond scission.We have also shown experimentally that the thiolate ligand control...

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Section: Coordination Structure Of Ferrous C357hmentioning

confidence: 96%

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam

Shiro¹,

Takahashi²,

Hori³

et al. 2001

Eur J Biochem

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“…2 c-h. Intriguingly, the frequency of the 4 band at 1,379 cm Ϫ1 of OS-II was even higher than that of ferric OxdA at 1,376 cm Ϫ1 (27). It has been established that the frequencies of the 4 band are influenced by the conjugative electron interaction between the central iron and the porphyrin ring, and that a decrease in the electron density causes an upshift of the 4 band (35). Thus, such a high 4 band frequency as 1,379 cm Ϫ1 is reminiscent of that of a ferryl-oxo heme species observed in horseradish peroxidase (called compound II), which exhibits a 4 band at 1,379 cm Ϫ1 (36).…”

mentioning

confidence: 99%

Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center

Konishi

Ohta²,

Oinuma³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…The Peak assignments were done according to earlier experimental and theoretical studies. 21,[35][36][37] asymmetric pyrrol half-ring vibration, producing the intensive SERRS peak at 1172 cm 21 (m 30 ), seen in Figures 1b and 2a, does not give any SERROA signal (Fig. 2b).…”

Section: Resultsmentioning

confidence: 94%

“…Assignments of the peaks in the RRS spectrum of Hb and their attributions to vibration of certain heme bonds and the sensitivity to ligands were done according to earlier studies. 21,[35][36][37] Analysis of RRS and SERRS spectra of the Hb shows that most of the peaks have the same positions, which indicates the absence of artifact peak shifts caused by Ag NPs. The reference spectrum of a mixture of Ag NP colloids and water only can also be seen in Figure 1b (grey), which shows clearly that the SERRS peaks are attributed to the Hb in the sample.…”

Section: Resultsmentioning

confidence: 96%

“…It is well known that vibrations of pyrrol bonds, especially pyrrol half-ring vibrations, are very sensitive to the redox status of the heme Fe ion, and to the presence of the sixth ligand (O 2 , NO, CO). 24,29,30,35 Hence, it is possible that any change in Hb saturation with O 2 will be manifested in changes in the intensity or the position of the m 4 peak. However, specific designed experiments are needed to verify the sensitivity of SERROA signal (over SERRS) to specific changes in Hb conformation and oxygenation.…”

Section: Resultsmentioning

confidence: 99%

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Novel chiroptical analysis of hemoglobin by surface enhanced resonance Raman optical activity spectroscopy

Brazhe

Sosnovtseva

et al. 2009

Chirality

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The metalloprotein hemoglobin (Hb) was studied using surface enhanced resonance Raman spectroscopy (SERRS) and surface enhanced resonance Raman optical activity (SERROA). The SERROA results are analyzed and compared with the SERRS, and the later to the resonance Raman (RRS) performed on Hb. The SERRS measurements careful optimization, with respect to the concentration and volume ratio of the analyte to colloids, enables for the first time SERROA of this molecule. We observed that the most intense SERROA signals were attributed the m 4 , m 20 , and m 21 vibrations, which are sensitive to the redox state of the heme's iron ion, and to the presence of its sixth site, bound to exogenous ligand; O 2 , NO or CO. However, in this study, the SERROA signals corresponding to these vibrations appear more sensitive to the Hb oxygen-binding properties than they appear in the SERRS or RRS. Moreover, the SER-ROA signal of Hb has successfully been monitored as a function of time, and was observed to be stable for 4-5 min. To our knowledge, the SERROA results of Hb, and its comparison to SERRS and RRS, are here reported for the first time.

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Nature of the iron-ligand bond in ferrous low spin hemoproteins studied by resonance Raman scattering

Cited by 163 publications

References 7 publications

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam

Proximal cysteine residue is essential for the enzymatic activities of cytochrome P450cam

Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center

Novel chiroptical analysis of hemoglobin by surface enhanced resonance Raman optical activity spectroscopy

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