Heme Coordination Structure Research Articles

Identification of Nitric Oxide Synthase as a Thiolate-ligated Heme Protein Using Magnetic Circular Dichroism Spectroscopy: COMPARISON WITH CYTOCHROME P-450-CAM AND CHLOROPEROXIDASE

Nitric oxide (NO) has recently been recognized as an important biomolecule playing diverse physiological roles. It is synthesized in several different tissues from L-Arg and O2, using NADPH as an electron donor, by a family of heme-containing catalytically self-sufficient monooxygenases known as nitric oxide synthases (NOS). Recently, the CO complex of reduced NOS has been shown to exhibit an absorption maximum near 450 nm, a characteristic spectral feature of cytochrome P-450 (P-450). Yet, the amino acid sequences of NOS and P-450 have no homology. To further probe the active site heme coordination structure and the heme environment of NOS, we have employed magnetic circular dichroism (MCD) and CD spectroscopy in the present study. MCD spectra of several derivatives of rat brain neuronal NOS strikingly resemble those of analogous derivatives of bacterial P-450-CAM and fungal chloroperoxidase, two known thiolate-ligated heme proteins. Given the proven fingerprinting capability of MCD spectroscopy, this provides convincing evidence for endogenous thiolate (cysteinate) ligation to the heme iron of NOS. Furthermore, the heme-related Soret CD bands of NOS (positive) and P-450s (negative), as represented by P-450-CAM, are almost mirror images, whereas chloroperoxidase exhibits totally different CD band shapes. This suggests that the active sites of NOS and P-450 may share some common structural features, but significant distinctions exist between their heme environments in certain aspects such as hydrophobicity or size.

Heme coordination of prostaglandin H synthase.

The heme coordination of ovine prostaglandin H synthase (PGHS) has been characterized by EPR, magnetic circular dichroism, resonance Raman, and optical spectroscopies. The EPR spectrum of ferric PGHS is consistent with an equilibrium mixture of high-spin and low-spin heme species. Both species disappear on reaction of the synthase with hydroperoxides. The high-spin to low-spin interconversion is temperature- and concentration-dependent. Correlation between the axial and rhombic ligand fields of the low-spin heme species suggests that it has bishistidine axial ligation. Magnetic circular dichroism spectra of PGHS also show a temperature-dependent spin transition. Resonance Raman spectra indicate that the enzyme exists as a mixture of six-coordinate low-spin and six-coordinate high-spin ferric heme species. No Raman bands attributable to five-coordinate high-spin heme species are detectable. The magnetic circular dichroism spectra of the fluoride, azide, cyanide, and imidazole derivatives of PGHS resemble those of the corresponding metmyoglobin derivatives and are very different from those of the catalase derivatives. EPR spectra of the imidazole derivative of these three proteins provide additional evidence that the heme coordination structure of PGHS is similar to that of metmyoglobin rather than that of catalase. The midpoint potential of the PGHS Fe(III)/Fe(II) pair is in the range observed for hemeproteins with mono- or bishistidine coordination. These data provide a convincing case that the axial heme ligands of PGHS-1 are a pair of histidine residues, with the distal histidine weakly associated and possibly exchangeable with a weak-field ligand.

Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate ( l-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals ( g ⊥ = 6, g | = 2) along with weak low-spin signals ( g = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals ( g = 2.53, 2.18,, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.

The active sites of chloroperoxidase and cytochrome P-450-CAM: Comparative spectroscopic and ligand binding properties

Spectral similarities between chloroperoxidase (CPO) and cytochrome P-450 (P-450) have previously been used to suggest endogenous thiolate axial ligation to the CPO heme iron [1–6] as has been established for P-450. Both enzymes exhibit unique hyperporphyrin (‘split Soret’) spectra in their ferrous CO forms [1]. However, no free sulfhydryl group available for ligation to the heme iron have been found in chemical studies of CPO [7]. The t001. Soret Absorption Maxima (nm) and Spin States of the Ligand Complexes of Ferric CPO and P-450-CAM.aLigandCPObP-450-CAMcλ(ϵmM)Spin Statedλ(ϵmM)Spin StatedSH−449(e)ls467(43)flCH3COS−446(62)gls459elsCN−439(92)ls439(78)lsPyridine439(82)l421(111)lsNO437(114)ls430.5(105)lsCH3(CH2)3NC435(105)ls429.5(98)lsN−3432(110)ls427(81)lsSeCN−432(80)ls425(90)lsSCN−429(112)ls425(98)lsImidazole429(100)ls425(98)lsNo−2427.5(101)lseOCN−∼427els418.5(98)lsHCOO−425(121)ls419(106)lsHOCH2CH2SH418(69)ms464(58)fls417 (∼60)ms417(∼60)CH3CH2CH2SH416(74)ms464(∼33)flsCH3SCH3416(93)ms424(91)lsThioxane416(88)ms418(97)lsCH3SSCH3416(93)ms418(98)lsCH3COO−413(97)ms420(98)lsF−409(98)ghsno bindinga0.1 M K+ Phospate, 4 °C.bpH 6.0.cpH 7.0, (Refs.9–11).dls, low spin; ms, high-low mixed spin; hs, high spin.eUnstable.fRed Soret of hyperporphyrin spectrum (Ref. 11).gpH 3.0.t002. Soret Absorption Maxima (nm) of the Ligand Complexes of Ferrous CPO and P-450-CAM.LigandCPOa2 λ(ϵmM)P-450-CAMb2 λ(ϵmM)Me2PhPc2459d2460(98)CN−454(∼140)Not observedCH3(CH2)3NC452(141)452(106)CO444(167)446(120)NO440(114)438(83)a2pH 6.0.b2pH 7.0.c2Dimenthyllphenylphosphine.d2Not determined. MCD [4] and EPR [6] spectroscopic properties of analogous CPO and P-450 derivatives are generally similar, although some differences do exist. To obtain additional information about the active site structure of CPO, we have carried out extensive optical and MCD spectroscopic studies of both ferric and ferrous CPO in the presence of various exogenous ligands. The results obtained with CPO have been compared with those obtained with P-450 [9 - 11, this work] in order to obtain a better understanding of the electronic structure of the heme iron in both proteins.