Proc. Natl. Acad. Sci. USAVol. 75, No. 1, pp. 21-25, January 1978Chemistry

On the photosensitivity of liganded hemoproteins and theirmetal-substituted analogues

(metalloporphyrin/quantum yield/flash photolysis)

BRIAN M. HOFFMANt AND QUENTIN H. GIBSONtt Department of Chemistry, Northwestern University, Evanston, Illinois 60201; and t Section of Biochemistry and Molecular Biology, Cornell University,Ithaca, New York 14853

Communicated by Gregorio Weber, October 31, 1977

ABSTRACT We have examined the photosensitivity oflow-spin liganded hemoglobin, myoglobin, and peroxidase, andtheir metal-substituted analogues, using three different metals(Fe, Mn, Co) in several oxidation states and employing a varietyof diatomic or pseudo-diatomic ligands (L). We have discovereda number of photosensitive systems, and present an overallstereo-electronic classification scheme for these photodisso-ciation reactions: Linear, formally d6, metal-ligand fragments[e.g., Fe(II) + CO; Mn(II) + NO] are relatively photolabile, butsystems with a bent fragment, and higher electron occupancy[e.g., Fe(II) + 02; Co(II) + NO] are relatively photoinert. Pho-tostability appears to correlate with the occurrence of long-wavelength features in the optical absorption spectra, and theclassification scheme is explained by considerations of elec-tronic structure. The discussions are further applied to d5 sys-tems and to low-spin d6 metalloporphyrins with nitrogenousbases as axial ligands.

The photodissociation of CO from the carboxyferroheme ofcarboxyhemoglobin (HbCO) has been known since the workof Haldane and Lorrain Smith, 80 years ago (1). Sixty yearslater, Keilin and Hartree reported the ready photodissociationof CN- from ferroperoxidase-cyanide and ferromyoblobin-cyanide (2), and Gibson and Ainsworth extended the list ofphotodissociable ligands to include isocyanides, 02, and NO (3).This photolability of ferroheme complexes was contrasted withthe photoinertness of liganded ferrihemes, but it has not beenexplicitly recognized that the reported ferroheme photodisso-ciation quantum yields (<0) for ligand L fall into two classes:e.g., CO is highly photosensitive, with vco 1 (4, 5), compa-rable to values for simple metal carbonyls (see refs. 6 and 7), but02 and NO are minimally sensitive, with (PO2 - 10-2 and cONo

10-3. No satisfactory explanation of this variation has beenpresented, nor have the variations in photolability among li-ganded metalloporphyrins [MPor(L)] and among ligandedforms of hemoglobin [Hb(L)] and myoglobin [Mb(L)] beensystematically explored. In some cases, at least, a protein en-vironment appears to play a role; e.g., (PCO behaves differentlyfor MbCO and HbCO (4, 5), and isocyanide-hemes show a highquantum yield for photodissociation (-I) (Q. H. Gibson, un-published data) in contrast to a lowered value for isocyanide-hemoproteins (3). This has compounded the difficulty in iso-lating those factors that control the quantum yield for ligandphotorelease by a metalloporphyrin.

In our studies of metal-substituted hemoglobins (MHb) wenoted that MnHbNO and HbCO are isoelectronic, and weproceeded to confirm the resulting expectation that NO wouldbe readily photodissociated from the manganese protein, de-spite the minimal photolability of HbNO (8, 9). Encouraged

by this observation, we speculated that the variation in photo-dissociation quantum yields might be general, and might rep-resent a stereo-electronic discrimination in which MHb(L)systems with a linear, formally d6, metal-ligand fragment [e.g.,Fe(II) + CO; Mn(II) + NO] would be highly photodissociable,but systems with a bent fragment and higher electron occu-pancy [e.g., Fe(II) + 02; Fe(ii) + NO] would be relativelyphotoinert. This speculation has prompted us to undertake abroader study of the axial-ligand photodissociation from low-spin metalloporphyrins. We have primarily employed ligandedHb and Mb, and their metal-substituted analogues as a chem-ically flexible experimental system in which nitrogen atoms ofthe porphyrin and proximal imidazole occupy five coordinationsites of a first transition series metal ion, and the sixth site isoccupied by a reversibly bound diatomic or pseudodiatomicligand, L.We here discuss flash photolysis photodissociation mea-

surements for a large number of such systems, many previouslyunexamined. The results so far confirm the occurrence of adichotomy in the values of (0L, which we show to reflect thegeometry and electronic structure of the metal-ligand linkageand which appears to correlate with features of the opticalspectra.

MATERIALS AND METHODSHemoglobin and its liganded derivatives were prepared bypublished procedures, as were metal-substituted hemoglobinsand their derivatives (8, 9). Ligand photodissociation was ob-served spectrophotometrically after flash photolysis. Thesamples were exposed to light from a Multiblitz III electronicflash with 100-J energy input, and flash duration, (l/e) - 400Asec. The optical absorption spectra of low-spin liganded MPorand MHb and MMb are typically dominated by porphyrin-centered transitions, the Soret band in the vicinity of 400 nm(E of order 105), and the a-f bands in the vicinity of 550 nm (Eof order 104) (see refs. 10 and 11). The photoflash was typicallyscreened from the sample by a series of Corning glass filtersranging from yellow (3-72; 50% transmittance at X 455 nm)to red (2-62; 50% transmittance at X 608 nm), affording areproducible means of varying the effective numbers of quantadelivered into the a-f band of the samples, and at the sametime providing complementarity between an observing beamin the Soret region and the photoflash.

This technique assumes an approximate independence of q.from wavelength in the region of the metalloporphyrin a-flband, as has been found to obtain in earlier careful measure-ments of (oL(X) for heme-ligand photodissociation (ref. 5; Q.

Abbreviations: MHb, MMb, metal-M-substituted Hb and Mb; Por,porphyrin; TPP, tetraphenylporphinato; L, axial ligand; B, nitrogenousbase.

21

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22 Chemistry: Hoffman and Gibson

H. Gibson, unpublished data). As a check, the photoflash waslimited to the a-fl region by a 3-72 color filter and thenscreened by various neutral density filters, with equivalentresults. It was easily possible to make observations during theflash, which is important with ligands such as oxygen and nitricoxide. In the case of oxygen, for example, when hemoglobin isequilibrated within air, the rate of the recombination reactionis about 10,000/sec, which is sufficiently greater than the rateof decay of the photoflash so that the concentration of deoxyHbclosely followsthe flash profile.

In every case several concentrations of ligand were used to-gether within several levels of light intensity. Variation in ligandconcentration gives assurance that the observed rate is not de-termined by the photoflash, and variation of the light level, ifassociated within a corresponding change in extent of photo-decomposition, eliminates the possibility that an observed smallphotoreaction is due to full breakdown of a small amount of a

photosensitive contaminant, the main constituent being ef-fectively photoinsensitive. Finally, a check was made of thedirections of excursion after flashing, followed at severalwavelengths to confirm that these agreed qualitatively withindications from the absorption spectra of the derivatives beingexamined.The methods used do not lend themselves to the accurate

determination of quantum yields, because the overlap betweenthe photoflash and the a-fl absorption bands of the compoundsstudied will be somewhat different in each case. There is nodifficulty, however, in distinguishing between systems, suchas MbNO, in which sp is small, of order -10-3, and MbCO, inwhich (pco > 0.5 at room temperature. In the first case theobserved excursion drops steeply as the overlap between theexciting light and the absorption bands is decreased by insertingprogressively redder filters. In the second, there is virtually nochange in excursion until orange filters (e.g., Corning 3-68) are

used. Equivalently, reducing the flash intensity with neutraldensity filters, say, until 100% photolysis of MbCO ((pco - 1)is barely achieved, would give an undetectably small excursion

for MbNO. As presented below, all compounds newly examinedcould be assigned either to a low-sensitivity group (v < 10-3)or to a high-sensitivity group (ep> 0.1). In reporting new resultssome approximate numbers have been given; precise values of<(X) will be available from measurements employing a tunablepulsed-dye laser as excitation source.

RESULTS

Table 1 presents some representative values of pL. for a-f bandphotoexcitation of MMb(L) systems. In discussing the photo-reactivity of liganded metalloporphyrins we shall refer to thereactive fragment consisting of the metal-ion (M) plus disso-ciable ligand (L). We adopt the notation of Enemark and Fel-tham and denote this fragment by IMLIr, in which r is the totalnumber of electrons associated with the metal d and ligand7r*(L) orbitals (see ref. 13); L will be diatomic, AB, or pseu-

dodiatomic, and all systems considered are low spin.IML15. We recall that previous investigations have demon-

strated that low-spin ferriheme complexes appear to be com-

pletely photoinert (3).$ML16. It is well known that CO photodissociated from the

IFeCOi6 of MbCO, and that pco approaches unity. Equallyinteresting, the value of fpco for MbCO is wavelength inde-pendent from 280 to 546 nm (5), and probably at least as far tothe red as 620 nm (Q. H. Gibson, unpublished data). Theanalogous isocyanide complexes, fFeCN-RI6 are also photolabile(3); although (PCN-R varies with R, it is as high as 0.32 for the

Table 1. Representative ligand photodissociation quantum yields(n) for the IMLjr fragment of myoglobin and

metal-substituted myoglobins

r M L p. Ref.

6 Fe(II) CO 1 4,5Fe(II) CN- 1 2Fe(II) R-NC* >0.1 3, 12; Q.H.G.Fe(III) NO This workMn(II) NO -1 This workCo(III)t CN- >0.2 This work

7 Fe(II) NO <10-3 38 Fe(II) 02 <10-2 3

Fe(II) Phenyl-NO <10-4 Q.H.G.Co(II) NO <10-4 This work

9 Co(II) 02 <10-4 This work

Q.H.G., unpublished data of Q. H. Gibson.* Dependent on R; see text.t Co(III)-horseradish peroxidase was used instead of Co(III)Mb.

n-butylisocyanide complex of Mb (12). It is less well known thatferrous Mb, peroxidase, and Hb react reversibly with CN- andthat the IFeCN-I6 fragment is photosensitive, with 4PCN- t

(2).It is also useful to consider the properties of solution metal-

loporphyrins, although these typically constitute a less well-defined system than the MMb(L) because they potentiallypossess two dissociable axial ligands. Upon irradiation of car-bonyl-ferroporphyrin complexes with a nitrogenous base assixth axial ligand, the CO is lost with fpco 1 to form thehigh-spin five-coordinate ferroheme-base complex, in analogyto MbCO (14, 15). In addition, the photoreactivity of the low-spin (ferroheme)(L)2 complexes, in which L = R-NC, and CN-has been studied and in both cases the loss of one ligand, andpossibly its replacement by an H20, proceeds with a quantumyield of essentially unity. Thus, the available data for solutioncomplexes further indicate the ready photodissociability ofjMLj6 metalloporphyrin systems (16).

Interestingly, further irradiation of the (ferroheme)(L)(H20)complexes demonstrates the possible complexities associatedwith a mixed-ligand solution porphyrin system. The quantumyield for L = CO is also roughly unity, but for L = R-NC orCN-, sp < 10-4. In these latter cases (16), it may be that thephotorelease and rapid recapture of a trans H20 is occurringin preference to release of L.

As mentioned above, we earlier noted that the system [Fe(II)+ CO] is isoelectronic with [Mn(II) + NO] and anticipated thatthe binding properties should be similar. We proceeded todemonstrate that the {MnNO16 fragment of MnHbNO disso--ciates to Mn(II) and NO upon irradiation (8, 9), and modelcompounds confirm that in both systems the diatomic ligandbinds in a linear fashion (see ref. 17). Indeed we now find that~PNO - 1.

As a similar case of a d5 metal that binds NO to give anIMNO06 fragment, the ferric forms of hemoglobin, myoglobin,and horseradish peroxidase all bind nitric oxide; with these NOcomplexes being particularly stable in the latter two cases (18).We have prepared the nitrosyl complex of metmyoglobin, andin contrast to other complexes, flash photolysis experimentsshow that the bound NO is readily photodissociated ((Po0.5).

Finally, the cyanide complexes of met-cobaltmyoglobin andmet-cobalt horseradish peroxidase, with the ICo(III)CN-16fragment, are also reasonably photodissociable, with (PCN- >0.2.

Proc. Natl. Acad. Sci. USA 75 (1978)

Proc. Natl. Acad. Sci. USA 75 (1978) 23

{MLJ r7.7 In contrast to the highly photolabile system withr = 6, the complexes HbO2, with the fFeO218 fragment, andHbNO, with fFeNOJ7, are relatively photostable, with po < 10-2and 10-3, respectively. In addition, the aromatic nitroso com-plexes of Hb, which would be viewed as analogous of HbO2 andthus as having $MLJ8, are at least as photoinert (Q. H. Gibson,unpublished data). We have also examined the liganded cob-oglobins, both CoHbNO, fCoNOJ8, and CoHbO2, )C002J9, andfind that both have quite small values, sp < 1O-4. Interestingly,a low-temperature photolysis study found that CoHbO2 is lessphotosensitive than CoMbO2, apparently another instance ofprotein modification of (pL (19). This work, although non-quantitative, also suggests that Soret excitation of the oxy-cobaltmesoporphyrin-substituted Mb gives an unusually high quan-tum yield.No previous photolysis studies of metalloporphyrin solutions

with r > 7 have been reported, so we have examined the r =7 five-coordinate nitrosyltetraphenylporphinatoiron(II),Fe(TPP)(NO), and six-coordinate Fe(TPP)(pyridine)(NO) andfind both to be minimally photolabile. In addition, a low-temperature photolysis study also found oxy-cobalt porphyrin(r = 9) to be photoinert. Thus, the general photostability of M-Llinkage with r > 7 appears well established.

DISCUSSIONThe results presented here have been obtained from systemsinvolving three different metals in several oxidation states andemploying a variety of diatomic or pseudodiatomic ligands,both charged and neutral. They seem to suggest the systematicoccurrence of a bimodal distribution in the MMb(L) andMHb(L) photodissociation quantum yields. The differencebetween an MPor(L) with, say, JFeCOj6 on one hand and{FeO218, or fFeNOJ7 on the other is not bond strength: (i) Theenergy avaiable from photoexcitation in the a-f band is, say,52 kcal/mol for X = 550 nm, far in excess of the Fe-L bondstrengths of about 20 kcal/mol or less as indicated by the en-thalpy of binding (20). (ii) The order of bond strengths, 02 <CO <NO, is not mirrored in the behavior of5p: pco>> poj,pNo.Thus, we must examine the properties to be expected of theexcited states of metalloporphyrins with IMLI fragments, as-suming that the ligand photolability of MMb(L) is primarilydetermined by the lowest lying excited state of the system (6,7). This photoactive state may be the direct product of photonabsorption, or may be populated by radiationless transitions;although it is quite possibly of different multiplicity than theground state, at present we shall when convenient ignore thequestion of spin, considering only orbital configurations.The obvious explanation for the photolability of MHb(L)

systems with r =6 parallels previous discussions of metal-ligandphotodissociations in less complex inorganic molecules (6, 7; ref.21 and references therein). Shank et al. (22) observed thatHbCO photodissociation occurs in less than 0.5 psec. On thebasis of calculations of Zerner et al. (23), they suggested thatan initial porphyrin (ir - ir*) excitation decays without emit-ting radiation to a lower state, corresponding to a ligand field,(4r I dom), excitation at the metal. This state is strongly a an-tibonding between Fe and CO, with reduced ir bonding as well,and would promptly dissociate.

This explanation, however, requires further discussion, be-cause neither experiment nor theory gives direct evidence ofany excited states below (ir-7r*). Optical absorption measure-ments now place the (d4 - dm2) excitation for MbO2 andMbCN- at considerably higher energies than the lowest (or- 7r*)state (24, 25), and this assignment is supported by more recent

celculations (26, 27). These results, however, are for theground-state geometry of the system. It is quite possible thatthe minimum energy of the ligand field excitation involvessignificant distortion§ and that, say, the (d,-dz2) triplet state inits relaxed geometry has crossed below (7r-7r*). In this event, adissociating (d,-d.2) level could be populated by radiationlessprocesses. Photosensitization studies may clarify this point.

In the absence of definitive information about low-lying,higher-multiplicity ligand-field states, we have been led tonotice a possible alternate mechanism: For MbCO, and fML16metalloporphyrins in general, photodissociation might in factoccur from the lowest (ir-r*) configurations, either directlyfrom the excited singlet or from the triplet state after inter-system crossing. First, this scheme is also in accord with thewavelength independence of L. (5; Q. H. Gibson, unpublisheddata) and the prompt CO dissociation found by Shank et al.(22). Second, a considerable stabilization of the M-L bond ap-parently arises from xr back-bonding to L from the (don dYZ)orbitals of M, with donation in turn into these d4 orbitals fromthe highest filled porphyrin 7r(ai., a2.) levels (23, 26, 27); ex-citation of an electron from ir(aI., a2.) to lr* should decreaseM-L back-bonding. It is important to realize that, say, theFe-CO bond energy as reflected by .AH for the association ofMb and CO is -21.4 kcal/mol, but that the binding free energyat 298 K is only -1.82 kcal/mol [standard state, I torr (133 Pa)](20). Thus, it would take only a minor decrease of bond strengthto leave the CO effectively unbound. 11

Finally, we note that low-spin ferroporphyrin(B)2 complexes,in which B is a nitrogenous base (pyridine, imidazole), are notobservably photosensitive (14), although such complexes haveoptical spectra and orbital patterns similar to those for the r =6 pseudodiatomic adducts (18, 31) (Fig. 1). This difference isrationalizable if a dissociating triplet ligand-field state lies above(7r-7r*) in the former systems, but below it in the latter. How-ever, this difference might also be rationalized by the suggestionthat (7r-1r*) is the photoactive state in all these compounds.Unlike ir-acceptor ligands such as CO, nitrogenous ligands actprimarily as a donors. Indeed, the recent observation that themonoadduct Fe(TPP)(CO) is low-spin, whereas the Fe(TPP)(B)complexes are thought to be high spin, has been attributed tothe enhanced 7r-accepting characteristics of CO as comparedto base B (32). If ir back-bonding does not contribute to the Fe-Bbond strength, then the above discussion indicates that theporphyrin (7rf*ir*) excitation should not cause the photola-bilization of B.Why do the systems with r > 7 exhibit reduced photosensi-

tivity? One "trivial" answer would be the rapid recombinationof M and L following photodissociation, but that is ruled out,at least for HbO2 and MbO2. Within less than 0.5 psec afterexcitation of HbO2, Shank et al. observed an absorbance thatdecayed with a decay constant of -2.5 psec (22). Because thisobservation is independent of 02 pressure, they argued that thetransient is due to absorption by a short-lived excited state,rather than to an unobservably rapid 02 dissociation followedby rapid recombination. An even more compelling argumentagainst rapid recombination comes from the work of Austin etal. (33). They find that the maximum rate of 02 recombinationwith MbO2 (kba in their notation) at 295 K is spread around a

§ H. B. Gray has pointed out to us that this type of situation occurs inthe case of the [Co(CN)6J3- anion (28).This mechanism had been noticed previously by M. W. Makinen(personal communication).Because CO is bound much more tightly to Ru(Por) than to Fe(Por),it might be that either mechanism could also rationalize the low (Pofor Ru(Por)(CO)(B) (refs. 29 and 30).

Chemistry: Hoffman and Gibson

24 Chemistry: Hoffman and Gibson

xy -@*

xz,yz 4.

d(M) and 7r*(L)(r =6)

x2 _y2Z -7r L)

r* (e

* 7r(a2,) 7r'x(L)+z24.- ir(a ) 4

xzyz4 Z0

Porphyri n7r and 7r*

d(M) and 7n*(L)(r =8)

FIG. 1. The pattern of relevant porphyrin xr orbitals is given inthe center. The general pattern of d(M) and ir*(L) orbitals for r = 6is indicated at left and follows the extended Huckel calculation ofKirchner and Loew (26) and Eaton et al. (27) in placing the unfilledorbitals above 7r*(eg) (calculations in ref. 27 actually indicate that Z2and x2 y2 should be interchanged). The pattern of d(M) and 7r*(L)for r = 8, schematized at left, follows the scheme presented in ref. 32for convenience in correlating r = 8 with r = 6. Although extendedHuckel calculations give results quite different in detail, they confirmthe occurrence of an unfilled antibonding orbital lying below lr* (eg)(ref. 27).

most probable value of -107 sec-1, far too slow to be observedon the time scale of several psec.

Another interpretation of the behavior of the bent (13, 17,34, 35) r 2 7 linkage might be that the metal-ligand bondstrength depends less on ir back-bonding, and thus that theporphyrin (ir -.ir*) excitation affects the bond strength less.However, even the bent M-02 structure is found to exhibitconsiderable ir back-bonding (26, 27, 31). Rather, on both ex-

perimental and theoretical grounds, we here suggest as a more

satisfactory explanation that in these cases there does exist a statethat is lower in energy than (r-7r*), that is rapidly populatedby radiationless processes following photon absorption, and inwhich the M-L bond strength is either undiminished or reducedby a significantly smaller amount.

In systems with r > 7, theoretical analyses uniformly indicatethat the xr*(L) orbital of L lies below the 7r*(eg) porphyrin or-bital and that a [MPor -xr*(L)] state lies below the porphyrin(7r7r*) excitation (23, 26, 27, 34, 35); this is the reverse of thesituation in an r = 6 complex (Fig. 1). The existence of suchstates is borne out by optical spectroscopy. MHb(L) andMMb(L) with r > 7 have been found to exhibit near-infraredbands, in contrast with the r = 6 systems (10, 11, 24, 25), andfor MbO2, polarized single-crystal absorption measurementshave in fact led to the charge-transfer state assignment (24). Insuch a charge-transfer state the increased charge separationshould enhance ionic bonding, and it has indeed been shownin simpler Werner-type complexes that such states are relativelystable (36, 37,tt). Moreover, the low energy of these states shouldenhance their non-radiative decay, further minimizing pho-tolability (38).On the basis of these considerations, and the results for HbO2

(22), and MbO2 (31), we suggest that the low photodissociationquantum yields for $MLJr, r > 7, be interpreted in terms of aninitial (ir --ir*) excitation followed by a rapid (<0.5 psec) ra-

tt Note that additional nondissociating low-lying states are obviouslyavailable for r = 7 (Fig. 1).

diationless decay to a short-lived (decay constant, -2.5 psec forHbO2) relatively stable charge-transfer state. For the low-spinferriheme complexes (r = 5) with an unfilled set of d(t2) or-bitals, low-energy porphyrin -- metal charge transfer states

must exist (22), and similar arguments should thereforeapply.

In this work we have discovered an appreciable number ofphotosensitive metalloporphyrin systems. The results have ledus to a stereoelectronic classification scheme and an initial ra-tionalization for the photodissociation reactions of ligandedhemoproteins and their metal-substituted analogues. Theclassification is, of course, merely a first approximation. Forexample, as noted above, different M-L systems within a classhave different quantum yields, and M. for a given ligand candepend significantly upon its protein environment. Thus, fur-ther experiments may well show a more nearly continuousrange of pL. However, because we observe a correlation be-tween photodissociation quantum yields and the optical ab-sorption spectra, and because both are sensitive to the proteinenvironment, our observations open the possibility of combiningthese two tools in studies of the details of the electronic structureof a liganded metalloporphyrin and of their modification bya surrounding protein.

This work was supported by the National Institutes of Health, GrantsHL-1351 and GM-14276, and by the National Science Foundation,Grant BMS-00478. B.M.H. was the beneficiary of stimulating discus-sions with Profs. K. G. Spears, M. Makinen, H. B. Gray, and Dr. L. K.Hanson.

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7r*(L) - -z2

Proc. Natl. Acad. Sci. USA 75 (1978)

7r,, (L )

Chemistry: Hoffman and Gibson

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