Phorochem. Phorobiol. Vol. 35, pp. 751 to 759, 1982 Printed in Great Britain. All rights reserved

TECHNICAL NOTE

003 1-8655/82/050757-03S03.00/0 Copyright 0 1982 Pergarnon Press Ltd

A CONVENIENT PHOTOCHEMICAL METHOD FOR REDUCTION OF FERRIC HEMES

BRIAN WARD and C. K. CHANG Department of Chemistry, Michigan State University, E. Lansing, MI 48824, USA

(Received 15 October 1981; accepted 1 December 1981)

Abstract-A method has been developed for the reduction of ferric porphyrins and hemoproteins using ketyl radicals generated from ketone photoreduction. This method is applicable to both aqueous and non-aqueous systems and has several advantages over the dithionite reducing agent.

INTRODUCTION

Ferric hemes and hemoproteins in dilute solution have been shown to be reduced to the ferrous form by r-hydroxyalkyl (ketyl) radicals generated by pulse radiolysis (Brault et a/., 1980; Schafferman et al., 1974; van Leeuwen et a/., 1979; Goff and Simic 1975; Simic et al., 1975; Simic and Taub, 1978). The ketyl radicals, being strong reducing agents and soluble in organic solvents, compare favorably with conven- tional heme reducing agents such as aqueous dithio- nite. However, the method by which the ketyl radicals are generated is not easily adaptable to routine use. We wish to report a method based on radical oxi- dation by ferric heme during the course of the well- known ketone photoreduction (Neckers, 1967). Using this technique one can easily and cleanly reduce ferric hemes to the ferrous form even in anhydrous organic solvents without introducing extra (contaminating) ligands.

MATERIALS AND METHODS

Solcents. Toluene was purified by stirring with several changes of concentrated sulfuric acid followed by distilla- tion from lithium aluminum hydride. Tetrahydrofuran (THF)’ was distilled from lithium aluminum hydride. Pyr- idine was distilled from calcium hydride.

Hemes. Etioporphyrin and etiochlorin were prepared by standard procedures (Fuhrhop and Smith, 1975). Iron insertion was accomplished by the FeSO, method (Fuhr- hop and Smith, 1975). Horse heart myoglobin (Sigma. type 111) was used as received.

Photureducrion in non-aqueous systems. Ferric hemes either in the chloride or p o x 0 dimer form ( - 10 pM) and benzophenone ( - 100 pM) were dissolved in dry toluene, THF, or 0.2 M pyridine/toluene mixture. The solution was rendered oxygen-free by either flushing with Ar gas for 30min using a syringe needle or by freeze-thaw cycles under vacuum. The deoxygenated solution was then irra- diated with a Hg lamp (Hanovia ‘Utility’ UV lamp, 140 W). In general, reduction was complete within 20s. Spectral

* Abbreviations: Hb, hemoglobin; Mb, myoglobin; THF, tetrahydrofuran; the term heme is used nonspecifically to include various iron porphyrins.

. .

changes were monitored using a Cary 219 spectropho- tometer. In ail experiments further irradiation after the heme reduction was complete resulted in no spectral change.

Photoreduction in aqueous system. Myogiobin was dis- solved in 0.1 M potassium phosphate buffer (pH 7.0) con- taining 2% isopropanol and 0.008% acetophenone. The sol- ution was degassed and irradiated as above.

RESULTS

Figures 1A and B show the progress of reduction with Fe(II1) etioporphyrin chloride in toluene and THF, respectively. The final spectrum in Fig. 1A exhi- bits the unique multiple peaks in the soret region, characteristic of an uncoordinated (4-coordinate) fer- rous heme XBrault and Rougee, 1973). Upon addition of CO, the corresponding mono-CO and his-CO complexes can be observed. The reduced heme in T H F is the high-spin bis-THF adducts (Dolphin et a/., 1976; Reed et al., 1980).

Figure 2 shows the photolytic reduction of (Fe etiochIorin)20 in 0.2 M pyridine/toluene. The result- ing bispyridine hemochrome has a spectrum identical with that obtained by reduction of the ferric chlorin in pyridine with aqueous dithionite or hydrazine hy- drate.

The photoreduction of horse heart myoglobin is , illustrated in Fig. 3. Oxygenation of the reduced form

yielded stable oxy-Mb; addition of C O then readily displaced Olr in accordance with the known reactions of ferrous myoglobin.

DISCUSSION

The photoreduction of ferric heme is represented by Scheme 1, which shows the photochemical reduction of ketones (Neckers, 1967) followed by Fe(II1)- mediated radical oxidation (Brault er al., 1980; Schaf- ferman er a/., 1974; van Leeuwen et a/., 1979; Goff and Simic, 1975; Simic et al., 1975; Simic and Taub, 1977; Simic and Taub, 1978; Castro et al., 1974).

757

758 BKIAN WARD and C. K . CHANG

8 s e 5: a n

1 , , A Fe Etioporphyrin

(in toluene)

I

400 500 600 700

X, nrn

Figure I . Photoreduction of Fe(1ll)etioporphyrin I-chlor- ide. ( A ) In toluene solution containing 0.1 mM benzo- phenone. total irradiation time = 0. 3. 1 0 s for the traces. respectively. The arrows indicate the progression of reduc- tion. (B1 In tetrahydrofuran solution containing 0.1 mM

benzophenone. total irradiation = 0 and 5 s.

While both R and the ketyl radical seem capable of reducing Fe(I1I). examination of the redox potentials (Table 1) suggests that only those radicals which oxi- dize at more negative potentials than the Fe(l1); Fe(1II) couple (0 - 400 mV) would be the effective reducing species. Indeed. gas chromatographic analyses of the photoreaction products in our hemin chloride benzophenone/toluene system revealed no trace of benzyl chloride but only the dibenzyl (@CH2CH2@) coupling product. indicating that the benzyl radical did not participate in the reduction of hemin chloride.

0 OH I

4-C-R' -k R-H - 4-C-R' + R'

n Fe(II1) Fe(1II

R' = 4. CH, RH and R as shown in Table 1

Scheme 1

Heme photoreduction has previously been ob- served for a number of proteins. Among these are: cytochrome oxidase (Kitagawa and Orii, 1978: Adar and Yonetani. 1978: Salmeen er a/.. 1978). horse heart

Fe Etiochlorin

400 500 600 700

X , nm

Figure 2. Photoreduction of Fe(ll1)etiochlorin I p o x 0 dimer in 0.2 M pyridine-toluene with 0.2 mM benzophe-

none. total irradiation: 0. 5. 10. 20 s.

chrome h (PoK and Butler. 1975). T-state hemoglobin (Kitagawa and Nagai. 1979) and cytochrome c s 5 2 (Kitagawa et d., 1980). Furthermore, heme photore- duction has purposefully been demonstrated with cyt P450 using a proflavin/EDTA system (Lipscomb et 01.. 1976, 1978) and cyt c using flavin/EDTA (Schmidt and Butler. 1976). The reduction presumably involved the prior photoreduction of a chromophore (e.g. Ra- vin) followed by heme reduction (Adar and Yonetani. 1978: Kitagawa er a/.. 1980: Manabe and Poff, 1975). In fact cyt h and cyt c S S 2 d o not photoreduce in the absence of flavin (Manabe and Poff. 1979: Kitagawa er 01.. 1980). Therefore. the mechanism of hemopro- tein photoreduction apparently is akin to Scheme 1 . One common feature to all of the protein photoreduc- tions. as well as to our system. is a wavelength depen- dence. For instance, cytochrome c oxidase is not pho- toreduced with visible excitation (Bocain pt d.. 1979) but is reduced in the UV region (Adar and Yonetani.

I , I , ' !

Myoqlobin

1

::

5: a

P n

X , nm

Figure 3. Photoreduction of metmyoglobin in 0.1 M potas- sium phosphate' buffer (pH 7.0) containing 0.008",, aceto- phenone and 2"" isopropyl alcohol. total irradiation: 0. 5.

cytochrome c (Vorkink and Cusanovich, 1974). cyto- 10, 25 s.

Technical Note 759

Table 1. Oxidation potentials vs. SCE of organic free radicals in H,O

RH R' Ei,,(V) Reference

d 2 C O H - 1.242* Rao and Hayon (1974) d t O H ( C H 3 ) - 1.532' Rao and Hayon (1974) 4 C H I 4 -0.2t3 Bansal and Henglein

(1974) 4 C H 3

- -0.8:s Bansal er ul. (1974) 0. OH

O H O H

- 1.29* Rao and Hayon (19741 I

(CH,),C' I

(CH 3 ) 2 C-- H

* pH 7. t pH 14. $ pH independent. $estimated from polarograms in references.

1978: Kitagawa and Orii. 1978: Salmeen er d., 1978). cytochrome c photoreduces 8 times faster at 410nm than at 535 nm (Vorkink and Cusanovich. 1974), as well T-state Hb displays a photoreductive wavelength dependence (Kitagawa and Nagai. 1979). In our sys- tem. irradiation through pyrex vs. quartz results in a very sluggish reduction. attributable to the low absorptivity above 310 nm of benzophenone or aceto- phenone.

I: is also the experience of many workers in the field that ferric hemes in pyridine under C O atmos- phere can be photoreduced to the carbon monoxide complex. We have observed. however. that the rate of this reaction is related to pyridine purity. Using highly purified pyridine, this photoreduction. in fact. did not proceed to completion; but addition of trace amounts of &CO/i-PrOH resulted in complete reduction. Therefore, it seems there are impurity chro- mophores present in commercial grade pyridine which are responsible for initiating the photoreduc- tion.

In conclusion. we have developed a simple and re- liable technique for reducing ferric heme under a var- iety of conditions. This method is especially suitable for studies carried out in organic solvents as well as aqueous protein work. One advantage of this method is that tedious reductant titration may be avoided when excess reducing agent may be deleterious (i.e. O2 binding). The fact that the ketyl radicals are more powerful reducing agents than dithionite also suggests that some hemoproteins. e.g. cytochrome c3. which cannot be completely reduced by dithionite may do so with this technique.

Ac~~rion./c,tlyrrneiir.s-We thank Dr. P. J. Wagner and M. A. Meador for helpful comments. This work was supported in part by N S F (Grant CHE-7815285) and USDA (Grant 59-2261 -01-437-0).

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