BIOINORGANK!

CHEMISTRY

6,1-9

(1976)

1

Magnetic Circular Dichroism of Heme-Isocyanide Complex in Aqueous Media TORU

SHIMIZU,

TSUNENORZ

The Chemical Research Sendai, 980, Japan

Institute

NOZAWA,

and MASAHlRO

of Nonuqueous

Solutions.

HATANO

Tohoku

University,

ABSTRACT

The nature of the ethyl isocyanide compbxes of fen-i- and ferroheme was investigated iu terms of the magnetic circular dichroism @ICD) spectroscopy. The complex formation of heme with ethyl isocyanide was criticalIy dependent on the solvent system, thus, on structures of heme before addition of the Iigaud. The MCD spectra of the heme ethyl isocyanide complexes revealed that: (1) The addition of ethyl isocyanide to both ferri- and ferro-heme in an aqueous solution gave respective high-spin heme complexes with no more than one ethyl isocyauide molecrde per one heme. (2) The ferro-heme with ethy1 isocyanide iu an aqueous ethanol sohrtion formed a typical monomeric low spin ferro-hemochrome with two Iigauds (ethyl isocyanide) on both sides of the heme plane. (3) In the presence of excess ethyl isocyanide in an aqueous ethanoi solution the ferri-heme gave au MCD spectrum similar to that for the monomeric low spin ferro-hemochrome. Therefore, ethyl isocyanide was believed to reduce the ferri-heme in an aqueous ethanol solution. (4) The ferro-heme ethyl isocyanide compIex in an aqueous solution offered a Faraday B term around 450 nm instead of an apparent Faraday A term observed for the ethyl isocyanide complex of the cytochrome P450. These results are not consistent with the view that the ferro-heme ethyl isocyanide complex in au aqueous solution can be a heme chromophore model for the cytochrome P-450 ethyl isucyanide complex_

INTRODUCTION The carbon monoxide complex of the reduced cytochrome P-450 (P-450) has an absorption peak at 450 nm different from those of other b type hemoproteins with peaks around 420 nm [ 11. Many model studies have been carried out for this peculiarity as well as its interesting enzymic functions [ 2-7]Since the ethyl isocyanide-ferro-heme complex system shows an absorption peak at 455 nm similar to that for the ethyl isocyanide complex of P-450, it has been regarded as an ingeneous absorption spectral model for P-450 [2] _ The MCD has been shown to be more sensitive to structures and electronic @American ElsevierPublishingCompany, Inc-, 1976

2

T. SHIMIZU, T. NOZAWA AND M. HATANO

states of heme than the electronic absorption spectra [8-13]_ The MCD spectra can be separated into three components (A, B, and C terms) which have different origins [ 14-l 7) _ Faraday A term will be observed when the ground or excited state involves &me degeneracies. Faraday B term is nonzero only if the ground or excited state of a particuIar transition can mix with other excited states by the magnetic field. Faraday C term is due to the ground state degeneracy [ 14-17]_ Therefore, it was felt to be significant to investigate the nature of heme ethyl isocyanide compIex by use of the MCD spectroscopy, and to examine its propriety as a P-450 modeL Recently, the native cytochrome P-450 has been studied with MCD and revealed much information which could not be obtained by the electronic absorption spectroscopy especially in the oxidation and spin states [ IO-I3 ] _ Hence, we examined whether the heme-isocyanide complex is a good model for cytochrome P-450 or not, by means of MCD.

EXPERIMENTAL Mater& Co., Ltd. Ethyl isocyanide Hemin was supplied from Daiichi-Pharmaceutical was synthesized by the method of Jackson and McKusic [14]_ The other compounds used in this investigation were ail commercially available guaranteed grade reagent and were used without further purification.

Preparation

of Samples

A stock solution protohemin (1 mM) was freshly made up daily by dissolving hemin chloride in 0.1 N NaOH and used within the day of preparation. The stock solution was kept at 0°C during the measurement to avoid the spectral changes_ For the spectral measurement, the stock solution was diluted by LO times with 0.1 M potassium phosphate buffer. Reduction was affected by he addition of a slight excess of sodium dithionite under nitrogen atmosphere_

Method of Measurement Absorption spectra were obtained on a Hitac‘hi Model EPS-ST spectrometer, using cuvettes of 2- and I-mm optical paths at 25*C. MCD spectra were recorded on a JASCO Model J-20A spectropolarimeter equipped with a JASCO eIectromagnet to produce a longitudinal magnetic field at the sampIe up to 12.5 kG_ Sample temperature was kept at 25°C. Cuvettes with S- and 2-mm optical paths were used_ MCD is expressed in terms of molecular ellipticity on the basis of heme molar concentration;

MCD OF HEME-ISOCYANIDE

COMPLEX

3

FIG. 1. MCD (upper) and absorption (below) spectra of ferroheme (0.1 m&l) reduced by (left side) and sodium dithionite in water-ethanol (l:I, v/v) in the absence ( -) presence of 5.4% (w/w) (1.02 M) ethyl isocyanide ( - - - - ) (right side).

@I,

= 3300 X AeN

where AEM is the difference in the molar extinction coefficients for the left circularly polarized light from that for the right per unit magnetic fieId (Gauss).

RESULTS Figure 1 shows the MCD and absorption spectra for the ferro-heme in a water-ethanol solution (1: 1) v/v) in the absence (solid line) and presence (broken line) of ethyl isocyanide. The MCD spectrum of the ferro-heme with ethyl isocyanide exhibits three clear A terms associated with the Soret (around 425 nm), Qe-1 (around 535 nm) and Qo_0 (around 565 nm) absorption bands. This is characteristic feature for Dan metalloporphyrin, to which the ferro-heme with low spin iron [8,18,19], or zinc porphyrin [ 19-211 belongs. This indicates that

4

T. SHIMIZU, T_ NOZAWA AND M. HATANO St..,....,._..,.

..,ul

4 3 2

420

A

3: . _

1 c53,1 0 -3. -2

\/ V

-3 -4

i

-30

-i -40

-5

__. WAVELENETHH(

AQ )

FIG. 2. MCD

Magnetic circular dichroism of heme-isocyanide complex in aqueous media.

BIOINORGANK! CHEMISTRY 6,1-9 (1976) 1 Magnetic Circular Dichroism of Heme-Isocyanide Complex in Aqueous Media TORU SHIMIZU, TSUNENORZ The Chem...
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