Axial Coordination in Nickel Porphyrins and Nickel-Reconstituted

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Downloaded by UNIV OF MICHIGAN ANN ARBOR on November 13, 2014 | http://pubs.acs.org Publication Date: October 15, 1986 | doi: 10.1021/bk-1986-0321.ch016

Axial Coordination in Nickel Porphyrins and Nickel-Reconstituted Heme Proteins Investigated by Raman-Difference and Transient-Raman Spectroscopy 1

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J. A. Shelnutt , K. Alston , E. W. Findsen , M. R. Ondrias , and J. M. Rifkind 1

Sandia National Laboratories, Albuquerque, NM 87185 Benedict College, Columbia, SC 29204 Department of Chemistry, University of New Mexico, Albuquerque, NM 87131 National Institute on Aging, National Institutes of Health, Baltimore, MD 21224

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Nickel-porphyrin complexes and Ni(II)-reconstituted hemoglobin ( Hb) and myoglobin ( Mb) have been investigated using cw and transient resonance Raman spectroscopy. The state of axial coordination at the metal in these materials can be determined using the characteristic frequencies of the Raman marker lines arising from the porphyrin moiety. The existence of only one axial ligand in the proteins is supported by identification of the axial ligand― Ni stretching vibration by isotopic substitution. The frequency of the Ni-ligand mode is consistent with histidine as the fifth ligand. The effects of the T->R structural change in the protein environment of the Ni porphyrin are observed in the Raman spectrum. T-R structural changes at the metal are associated with changes in the electronic structure of the porphyrin ring. We have also investigated the photoinitiated ligand-release and ligand-uptake processes in excited states of the Ni porphyrins and Ni-reconstituted proteins. The results indicate that the protein matrix effectively controls photoinduced ligation dynamics. Laser excitation of ~10-ns duration induces the release of axial ligands in coordinating solvents, but not for the histidine ligand of Ni-reconstituted hemoglobin and myoglobin. Ligand uptake is observed in Hb when the 4― coordinate sites are excited. Ni

Ni

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Nickel porphyrins are of i n t e r e s t because of t h e i r occurrance i n coal, shale, and petroleum deposits (1) and because of t h e i r key role i n b i o l o g i c a l conversion of COg t o methane (2-5). The enzyme methylreductase i s the nickel-tetrapyrrole-containing enzyme that catalyses the f i n a l step, and possibly other steps, i n the 6 electron reduction of COg to methane ( 6 ) . The active s i t e of the methylreductase enzyme contains a n i c k e l - s i r o c h l o r i n derivative called F43Q ( 6 - 8 ) .

Understanding the involvement of F 4 3 Q i n methane

production i s of importance t o the natural gas industry i o r 0097-6156/ 86/ 0321 -0232$06.00/ 0 © 1986 American Chemical Society

In Porphyrins; Gouterman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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u t i l i z i n g abundant inorganic resources f o r production of gaseous fuels. Recently, we have used resonance Raman spectroscopy to investigate a x i a l ligand processes at the metal i n Ni-porphyrin complexes and Ni-reconstituted heme proteins (9). These materials provide useful models f o r studies of a x i a l l i g a t i o n processes and for Ni-porphyrin-protein interactions that may be important i n the function of the methylreductase enzyme. Also, f o r the f i r s t time, we have used transient Raman spectroscopic techniques to examine the dynamical aspects of a x i a l l i g a t i o n at Ni porphyrins i n coordinating solvents (10) and i n the Ni-reconstituted heme proteins (11). The r e s u l t s of transient Raman investigations of the Ni-porphyrins, which show photoinitiated a x i a l ligand release and uptake, have important implications f o r f u e l generating photocycles based on these processes (12). Materials and Methods ^ H b and ^ N i - r e c o n s t i t u t e d Hb were prepared according to reported methods (13). Mb was made by the method of Alston and Storm (14) Solutions of the proteins i n 0.05 M phosphate buffer at pH 7.5 were used f o r obtaining spectra. The proteins are stable i n a i r and do not photodecompose. Ni protoporphyrin IX (Ni(ProtoP)), Ni uroporphyrin I (Ni(UroP)), and Ni octaethylporphyrin (Ni(OEP)) were obtained from Porphyrin Products and used without further p u r i f i c a t i o n . A l l solvents were of highest purity obtainable from commercial sources. A l l materials showed the l i t e r a t u r e u v - v i s i b l e absorption spectra. Absorption spectra were obtained on a PerkinElmer Model 330 spectrophotometer. Raman spectra were obtained on selected pairs of samples using a Raman difference spectrometer described previously (15). The_°/0° scattering geometry was employed. Spectral r e s o l u t i o n was 4 cm . A l l spectra were obtained at room temperature. Samples were excited with a krypton ion laser (Coherent 3000K) using the 415.1-, 413.1-, or 406.7-nm l a s e r l i n e s . To avoid heating, the samples were i r r a d i a t e d i n a r o t a t i n g (100 Hz) c e l l with two compartments. The laser power at the sample was less than 300 mW i n a p a r t i a l l y defocused beam. The Ni porphyrins are very stable to v i s i b l e l i g h t and even higher powers could be used. No sample decomposition was observed as determined by the lack of changes i n successive scans of the Raman spectrum during s i g n a l averaging and i n the absorption spectrum taken before and a f t e r the Raman spectrum. Nanosecond resonance Raman spectra were obtained using a nitrogen pumped dye laser (Molectron) and a spectrometer system described elsewhere (16). The ~180° backscattering geometry was used. "Low power" laser pulses ("0.3 mJ/pulse) s l i g h t l y focused with g c y l i n d r i c a l lens (f.1.= 250 mm) gave an energy density of ~ 5 mJ/cm . For "high power" spectra, the beam was t i g h t l y focused with a spherical lens ( f . l . = 100 mm); the photon density i s ~ 100 times greater than f o r the low power spectra. The pulse r e p e t i t i o n rate was 10 hz. S t a t i c cuvettes held the sample and the temperature was between 20-25 C. x


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P O R P H Y R I N S : E X C I T E D STATES A N D D Y N A M I C S

Results and Discussion Ni Ni State of A x i a l L i g a t i o n i n Hb and Mb. As we have previously reported, (17-20) a x i a l l i g a t i o n ^ a t thg metaj^ for non-hyper t r a n s i t i o n metal porphyrins (Zn , V=0 , Ni , Cu ) r e s u l t s i n a d i s t i n c t i v e pattern of s h i f t s i n the Raman e l e c t r o n i c structure marker l i n e s . The marker l i n e s are the oxidation-state marker l i n e (21-24) l/^ and the l i n e s s e n s i t i v e to porphyrin core size (centerto-nitrogen , distance) (24,25). The pattern of s h i f t s that indicates aJcïaï ligation i s characterized by (1) roughly comparable decreases i n frequency for a l l of the core-size marker l i n e s (including i/^, and i/^) and (2) r e l a t i v e l y smaller, but correlated, s n i f t s i n the oxidation-state marker l i n e l/^. Figure 1 shows the Raman spectrum of "^b obtained with 406.7and 413.1-nm e x c i t a t i o n and the spectrum of monomeric, fourcoordinate Ni protoporphyrin i n aqueous micellar s o l u t i o n (9). E x c i t a t i o n at 413.1 nm i s at resonance with the red component of the s p l i t Soret band of Ni-reconstituted hemoglobin; at 406.7 nm the blue component of the Soret band i s s e l e c t i v e l y probed. Comparison of the spectra shows that two sets of marker l i n e frequencies e x i s t . One set (labeled 4 i n Figure 1) i s enhanced by resonance with the blue Soret component; the other set (labeled 5) i s enhanced by e x c i t a t i o n of the red Soret component. Thus, the s h i f t s i n the core-size l i n e s i n going from set 4 -> 5 are -39 cm ( i / at 1657 cm ), -20 cm {y^ at 1593 cm ), and -34 cm {y^ at 1519 cm ). At the sametime tne s h i f t in-the oxidation-state marker l i n e i s only -11 cm {y^ at 1378 cm ). This pattern of s h i f t s i s consistent with a x i a l l i g a t i o n f o r the form whose Raman l i n e s are labeled 5 i n Figure 1. Comparison with a x i a l ligand complexes formed by Ni porphyrins i n coordinating solvents (e. g. p y r r o l i d i n e , piperidine, and pyridine), however, shows a clear d i s t i n c t i o n between the frequencies of the "form 5" marker l i n e s of the reconstituted proteins and those of the 6-coordinate models (9). The s h i f t s of the 6-coordinate species r e l a t i v e to the 4-coordinate species are larger than f o r the form labeled 5 (Figure 1) of the^proteins. The s h i f t s f o r the 6-coordjnate models are about -41 cm ( iq) > "^7 _^ cm (i/g) , and -41 cm (l/«) f o r the core-size markers ana -12 cm ( i ^ ) f o r the oxidation-state marker. No coordinating solvents give 6-coordinate complexes with frequencies close to those of form 5 of the protein. However, because the pattern of s h i f t s f o r form 5, r e l a t i v e to the 4-coordinate form, i s the pattern associated with a x i a l l i g a t i o n and because the marker frequencies of form 5 of the protein are d i s t i n c t from those of known 6-coordinate Ni porphyrins i n coordinating solvents, we propose that form 5 of the proteins i s a 5-coordinate species. |

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Ligation-induced Core Expansion. Based on the empirical relationships (given by Spiro (24)) between core size and Raman frequencies f o r the l i n e s and I / - Q and using the observed center-to-nitrogen distance of 2.038 Â l o r the Ni tetramethylpyridylporphine bis-imidazole complex (26), the predicted frequencies are: l/^, 1485; i> , 1560; I/^Q, 1615 cm . These predicted values compare favorably with experimental frequencies of 1475-1479 (i/g), 1560-1568 (v£, and 1615-1618 cm (i/- ) for a variety of 6-coordinate Ni-protoporphyrin complexes (Ôj. 2

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SHELNUTT ETAL.

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Figure 1. Raman spectra of nickel-reconstituted hemoglobin (pH 7.5) obtained with 413.1-nm excitation (a), with 406.7-nm e x c i t a t i o n (b), and the spectrum of n i c k e l protoporphyrin IX free acid i n cetyltrimethylammonium bromide micelles i n 0.1 M NaOH ( c ) . Spectra i n (b) and (c) were obtained simultaneously on a Raman difference spectrometer.


PORPHYRINS:

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E X C I T E D STATES A N D D Y N A M I C S

For the 5-coordinate species e x i s t i n g i n the proteins a smaller core s i z e of ~2.03 Â i s predicted on the basis of the frequency of J>2 and i/^q, but a s t i l l smaller core s i z e ~2.00 Â i s expected on the basis of I/g* The discrepancy may r e s u l t from d i s t o r t i o n of the porphyrin macrocycle i n the protein, n i c k e l being out-of-plane, or from peripheral substituent o r i e n t a t i o n a l e f f e c t s on v^. A x i a l Ligand-Ni Stretching Mode. Isotopic s u b s t i t u t i o n supports the i d e n t i f i c a t i o n ogâ 5-coordinate species i n the proteins. Substitution of N i into Hb r e s u l t s i n a 7-cm decrease i n frequency o| the l i n e at 236 cm" (9}- No other l i n e s between 200 and 500 cm s h i f t by more than 1 cm . A pure Ni-ligand mode would s h i f t by 10 cm . Thus, the mode at 236 cm i s about 70% pure. The Ni-ligand mode can be i d e n t i f i e d as the N i - a x i a l ligand stretch because of the close analogy between the Ni i s o t o p i c s u b s t i t u t i o n r e s u l t s and the Fe-isotope s u b s t i t u t i o n data f o r native hemoglobin and myoglobin (27-30). For the native proteins mjje ^ complete isotope s u b s t i t u t i o n studies wgye dgne, including N+ Ν s u b s t i t u t i o n of the pyrrole nitrogens, Fe+ r e s u b s t i t u t i o n , and p a r t i a l deuteration of the protein (30). On the basis of t h i s work and the d i r e c t correspondence between the Raman spectra of the N i r e c j n s t i t u t e d and native proteins, i t i s possible t o assign the 236cm l i n e to the N i - h i s t i d i n e s t r e t c h . H i s t i d i n e i s indicated as the a x i a l ligand by the frequency of the mode, which comes i n the region of F e - h i s t i d i n e stretching frequencies i n heme proteins and model 2-me^hylimidazole-heme complexes (190-275 cm ) (27-31). The f a c t that Hb has the normal Τ quaternary structure (9,13,32-36) also suggests that h i s t i d i n e i s the a x i a l ligand. The low frequency Raman spectrum of the 6-coordinate models i s d i s t i n c t l y d i f f e r e n t from the low frequency spectrum of the 5coordinate s i t e s of the Ni gjobins. In f a c t only one l i n e , a very weak shoulder on the 300-cm l i n e , i s observed i n the expected metal-ligand stretching region below 300 cm . F u r t h e r ^ n o predominately Ni stretches are found i n the 100-500-cm region. Instead, i n both the 6- and 5-coordinate cjmpjexes many of the low frequency Raman l i n e s show small (< ~1 cm ) Ni i s o t o p i c s h i f t s i n d i c a t i n g some N i - N p ^ character.


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Protein-Conformation E f f e c t s on the Metal-Protein Linkage. Since we have been unable to f i n d model Ni-porphyrin complexes whose Raman frequencies come close to those of the Ni-reconstituted proteins, i t i s c l e a r that the protein matrix has an impressive e f f e c t on a x i a l l i g a t i o n . We might also ask what i s the e f f e c t of changes i n protein conformation on a x i a l l i g a t i o n . Native i r o n hemoglobin e x i s t s i n a quaternary conformation which i s distinguished by i t s low Og a f f i n i t y . The low a f f i n i t y protein conformation, c a l l e d the Τ structure, has also been characterized by x-ray c r y s t a l structure (37,38) and many other physical techniques including resonance Raman spectroscopy (39,40). S p e c i f i c a l l y , the protein structure near the Fe porphyrin (heme) i n the Τ structure i s d i s t i n c t from that of hemoglobins with bound exogenous ligands. These liganded hemoglobins have a d i f f e r e n t quaternary conformation c a l l e d the R structure (38). Several deoxyhemoglobins and deoxymyoglobin also possess the R structure and they are characterized by high 0 a f f i n i t y . The Raman spectra of 9



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the R- and T-structure deoxyhemoglobins d i f f e r i n the frequency of the F e - h i s t i d i n e stretching v i b r a t i o n (39) and the^j-electron density marker l i n e s ^ ( 4 0 ) . Thus, a comparison of Hb which has the T-conformation and Mb which has an R-like structure can give some idea of the e f f e c t of a well defined protein conformational change at the^Ni-porphyrin s i t e . Hb possesses both 4- and 5-coordinate forms as demonstrated by the Raman spectra (Figure 1) and the s p ^ i t Soret band of the absorption spectrum (9,36). In contrast, Mb shows only the red Soret component and the Raman l i n e s c h a r a c t e r i s t i c of the 5coordinate form. Thus, myoglobin's R-like structure favors the 5coordinate form. The R/T difference i n a f f i n i t y f o r h i s t i d i n e might also be expected to reveal i t s e l f i n the strength of the N i h i s t i d i n e bond. In native Fe hemoglobin, the F e - h i s t i d i n e bond increases i n strength upon conversion from the Τ to R structure (31,39). To determine the R/T difference i n the N i - h i g t i d i n e force constant we must locate the N i r h i s t i d i n e mode i n Mb and compare^ the frequency with the 236-cm~ value previously determined f ^ r Hb ( 9 ) . ^ F i g u r e 2 shows the Raman spectrum of natural abundance Mb and Ni-reconstituted Mb i n the 100-500-cm" region. The spectra were obtained with 413.1-nm e x c i t a t i o n . The N i - h i s t i d i n e mode, which i n Mb i s almost degenerate with a strong mode of the porphyrin r i n g at 243 cm , cannot be resolved. However, the Raman difference spectrum i n d i c a t e ^ a decrease i n i n t e n s i t y near 241 cm and an increase near 233 cm" f o r the Ni-reconstituted myoglobin r e l a t i v e to the natural abundance protein. For 415.1-nm e x c i t a t i o n (spectrum not shown), the porphyrin mode i s apparently weaker r e l a t i v e to the N i - h i s t i d i n e mode. Even so, the N i - h i s t i d i n e mode i s not resolved i n the spectrum of the natural abundance protein. However, the large i s o t o p i c s h i f t i n the Ni protein reveals the mode as a c l e a r low frequency shoulder at about 233 cm i n the 415.1-nm spectrum. ^ In addition, another weak l i n e at "270gcm i n natural abundance Mb s h i f t s to ~260 cm" f o r the Ni protein (Figure 2 ) . The weak 270-cm" l i n e may represent a f r a c t i o n of the protein with a d i f f e r e n t state of a x i a l l i g a t i o n . A l t e r n a t i v e l y , another almost pure Ni-porphyrin v i b r a t i o n i s a p o s s i b i l i t y . Using the 241-cm value^for the N i - h i s t i d i n e frequency i n Mb, the T+R s h i f t from the R V M b comparison i s ~5 cm . This value i s consistent with the increase observed i n a comparison of the corresponding Fe proteins and with the T+R s h i f t s based on other Fe hemoglobins (3-8 cm" ) (31). The s i m i l a r i t y of the increases observed i n the metal-histiaTne frequencies f o r the n i c k e l and i r o n hemoglobins indicates that the e f f e c t of quaternary structure on the N i - h i s t i d i n e bond i s s i m i l a r to the Fe case. Also, the e f f e c t of the protein conformation change i s v i r t u r a l l y independent of the p a r t i c u l a r metal i n the porphyrin core. x

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Protein-Conformation E f f e c t s on E l e c t r o n i c Structure of the Porphyrin Ring. In s p i t e of the s i m i l a r i t y i n the R/T changes at the metal-histidine bond f o r Ni and Fe hemoglobins, the e f f e c t of the R/T conformational change on the porphyrin's e l e c t r o n i c structure i n the two cases i s quite d i f f e r e n t . In a wide v a r i e t y of hemoglobins the T+R increase i n J/(Fe-his) i s associated with a




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Figure 2. Raman spectra of Ni-reconstituted myoglobin and natural abundance Ni-reconstituted myoglobin and the difference spectrum. The Raman difference spectrum s h o w s j l e a r l y the isotope s h i f t i n the ~270-cm l i n e to ~260 cm and the larger (intensity-wise) s h i f t i n the N i - h i s t i d i n e mode at ~240 cm .


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decrease i n i / ^ . In contrast, ν* increases by 0.3 cm f o r the N i reconstituted proteins (T+R) (9J. The r e l a t i o n s h i p between i/^ and l/(Ni-his) i s more l i k e the l i n e a r c o r r e l a t i o n between these l i n e s seen f o r peroxidases (41-43) than f o r the a n t i - c o r r e l a t i o n observed for Fe hemoglobins (31). The difference i n the way the conformational change at the metal-histidine bond influences the porphyrin's e l e c t r o n i c structure ( i . e., the π-charge density, as measured by the oxidation-state marker l i n e ) f o r the Ni and Fe proteins, probably r e s u l t s from the differences i n the way each metal i n t e r a c t s with the porphyrin r i n g . The differences i n the metal-ring i n t e r a c t i o n i s partly a r e s u l t gf the d j j f e r g n t jj e l e c t r o n i c configurations of the metals (d f o r Ni ; d f o r Fe ) and the mixing of e (d ) metal o r b i t a l s with the e (ττ ) porphyrin o r b i t a l s i n the c a s l of Fe (44).


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Excited State A x i a l L i g a t i o n Processes i n Coordinating Solvents. When n i c k e l porphyrins are i r r a d i a t e d with ~10-ns laser pulses i n the 400-500-nm region, changes i n a x i a l coordination can be detected by changes i n the Raman marker l i n e s . The time evolution of a x i a l ligand complexes of Ni porphyrins has been f u l l y characterized by time-resolved absorption spectroscopy by Holten and coworkers (4547). Recently, we have obtained (10) the f i r s t Raman spectra of the transient Ni-porphyrin species observed by Holten. The Raman spectra p o t e n t i a l l y contain considerably more s t r u c t u r a l information than the absorption spectra. On the other hand, because of the weakness of the Raman s c a t t e r i n g i t i s more d i f f i c u l t to follow the time evolution of the transient species. The d i f f i c u l t y r e s u l t s from the neccessity of using an intense probe beam to excite the Raman spectrum as well as an intense pump pulse that generates the transient species at zero time. Unlike the weak probe pulse used i n time-resolved absorption spectroscopy, the intense probe pulse needed to excite the Raman spectrum may i n t e r f e r e with the evolving transient species. Nevertheless, one can usually i d e n t i f y the spectrum of the transient species superimposed on the spectrum of the s t a r t i n g material by comparing the spectra obtained with high and low power pump/probe pulses (Figures 3 and 4). E x c i t a t i o n wavelength dependence of the Raman l i n e s presents another complication that i s not a problem f o r transient absorption measurements. For example, i n comparing spectra taken at two d i f f e r e n t e x c i t a t i o n wavelengths, one must consider not only which and i n what proportion each molecular species i s pumped, but also keep i n mind what differences i n r e l a t i v e i n t e n s i t y of the Raman l i n e s r e s u l t from e x c i t a t i o n at the two e x c i t i n g wavelengths. The complication can usually be overcome, especially when the spectra of the two species are well resolved. We have previously investigated ligand release i n the 6coordinate piperidine complexes of Ni octaethylporphyrin (10). For Ni(OEP), formation of the 6-coordinate complex i s not complete, and so, the picture i s complicated by the presence of both 4- and 6coordinate species i n the i n i t i a l sample. However, upon e x c i t a t i o n with the pulsed laser the r e l a t i v e proportions of the two Ni(OEP) species change as determined by changes i n the r e l a t i v e i n t e n s i t i e s of the 4- and 6-coordinate sets of Raman marker l i n e s . Thus, a x i a l ligand release i s observed i n the excited state generated during the



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Ni(ProtoP)/pyrrolidine 420-nm excitation

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90% 6-coordinate form). The laser e x c i t a t i o n wavelength of 420 nm p r e f e r e n t i a l l y excites the 6-coordinate form which happens to dominate the absorption at t h i s wavelength (9-11). With respect to the low power spectrum, the spectrum obtained at high power shows increased i n t e n s i t y i n the l i n e s at 1660 (^(p » 1595 ( i / ) , and 1522 cm (l/^) . These l i n e s come from the 4coordinate form. Also, of t h j 6-coordinate form develops a high frequency shoulder near 1378 cm , the expected frequency of the 4coordinate species. The r e s u l t s c l e a r l y show that the piperidine ligands are l o s t i n the excited state. Thus, we f i n d that the Ni(ProtoP) case gives r e s u l t s s i m i l a r to Ni(OEP) i n p i p e r i d i n e . Photoinduced ligand d i s s o c i a t i o n can be understood i n terms of the ligand a f f i n i t y properties of the excited states of the metalloporphyrin (47). Ni porphyrins i n coordinating solvents display a ground-state equilibrium between the l i g a t e d Bstate with one electron each i n the metal d 2 2 and d^2 o r b i t a l s and the unligatedg A^ state with both electronsîn d 2 and none i n d 2_ 2. (In the d configuration of Ni(II) a l l other â o r b i t a l s are ^ completely f i l l e d . ^ For the 6-coordinate species, absorption of l i g h t by the j π t r a n s i t i o n s of the porphyrin r i n g generates the d i s s o c i a t i v e A^ d-d excited state (45-47^. The ground state of the liganded s j e e i e s i s the (d 2, d 2_ 2) state; the lowest excited state Ai s rapidly ^< 15 psJ reacnfd by nonradiative decay. Because èfie f i l l e d d 2 cannot act as a α-acceptor o r b i t a l for the a x i a l ligands, the ligands are l o s t u n t i l the excited state decays and ligands can be recovered. Rebinding of the ligands proceeds over an a c t i v a t i o n b a r r i e r and requires greater than 20 ns (45). Photoinduced ligand uptake also occurs (45-47). Unligated Ni porphyrins i n the equilibrium mixture of 4- and 6-coordinate species may be induced to bind ligands upon absorption of a photon. The 4coordinate Ni-porphyrin species are i n the Astate i n i t i a l l y . Upon photon absorption and decay of the π + π ^states the Ni porphyrin ends«up i n the lowest excited state which i n t h i s case i s the unligated B^ . The excited B^ state i s ^ a s s o c i a t i v e ; therefore, either^ligands are acquirid or the Bstate decays back to the unligated ground state. ^ We have observed the unligated Bexcited state i n noncoordinating solvents (11,45-47), buE not i n ones that coordinate. Using transient absorption spectroscopy, Holten and coworkers found that the l i f e t i m e of the Bstate f o r Ni porphyrins i n toluene i s ~250 ps- During our 10-ns pulse, a s u f f i c i e n t concentration of the Bexcited state can be generated to obtain the Raman spectrum. In p a r t i c u l a r , we have observed the unligated Β state f o r Ni(OEP) i n toluene, Ni(ProtoPDME) j n toluene and acetone, and Ni(ProtoP) i n micelles (11). The Bexcited state i s characterized by s h i f t s i n 1/^, and I/^Q t o frequencies that are intermediate between tê 4- and 5-coordinate values. For example, u of the unligated Bexcited state of Ni(ProtoP) and Ni(ProtoPDME) i s i n the range i§25-1632 cm for various solvent systems. For comparison, 5-coordinate Ni(ProtoP) i s i n the 1618-1621-cm range, while 4-coordinate frequencies are


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i n the 1652-1660-cm range. Thus, about three-fourths of the core expansion that occurs on formation of the 6-coordinate complex i s due to the d-d t r a n s i t i o n ; the remainder of the expansion r e s u l t s from adding the a x i a l ligands and the ligands' influence on the metal ^p^-^îlt) i n t e r a c t i o n (19). Ligation-Deligation Processes i n Nickel-Reconstituted Heme Proteins. In contrast with the 6-coordinate models, 5-coordinate s i t e s of Hb and Mb show v i r t u a l l y no difference between the Raman spectra obtained with low and high powers f o r excitation at 420 nm. The undetectably low quantum y i e l d f o r d i s s o c i a t i o n f o j the n i c k e l globins suggests that either (1) the d i s s o c i a t i v e A- state is^not reached (with s i g n i f i c a n t y i e l d ) after absorptioon by the π + π t r a n s i t i o n or (2) the h i s t i d i n e ligand i s regained rapidly on the nanosecond time scale of the Raman measurement because of cage (geminate) recombination. Rapid geminate recombination ( i . e. much f a s t e r than the 10-ns probe pulse) would insure that the steady state concentration of 4-coordinate species does not increase f o r high pulse power. For the 6-coordinate complexes we observe excited state ligand loss because the ligands are l o s t rapidly (~50 ps) and regained slowly with respect to our 10-ns pulse. Thus, during our pulse few deligated molecules regain ligands. Apparently, f o r the Ni globins the b a r r i e r to recombination i s d r a s t i c a l l y lowered by the protein matrix and recombination of the ligand i s much f a s t e r than i n the coordinating solvents. The absence of ligand release i n the n i c k e l globins i s probably not the r e s u l t of a change i n the decay pathway. The same t r a n s i t i o n s are expected i n the region between the π •> π r i n g t r a n s i t i o n s f o r 5- and 6-coordinate Ni porphyrins (45,48); therefore, the low y i e l d of photodissociation i s not a r e s u l t of additional decay pathways f o r the 5-coordinate species. Second, we also do not expect that the low l y i n g d •+ d t r a n s i t i o n moves above the π + π states f o r the 5-coordinate form (48). Consequently, there are no alternative or new branching pathways f o r decay of π + π excited 5-coordinate Ni porphyrins and the d d dissociative state remains the primary decay route. Thus, the most reasonable explanation f o r the lack of differences i n the high and low power spectra of the Ni-reconstituted globins when the 5-coordinate s i t e s are p r e f e r e n t i a l l y excited i s based on rapid geminate recombination rather than f a s t e r or altered decay mechanisms. The n i c k e l proteins and the native iron globins show the same behavior i n regard to f i f t h - l i g a n d photodissociation but f o r t o t a l l y d i f f e r e n t reasons. Heme proteins and other metal-reconstituted heme proteins have been investigated by transient absorption spectroscopy (49-53) and transient Raman spectroscopy (16,54-62). In none of the Fe proteins i s loss of the h i s t i d i n e ligand observed even on a picosecond timescale. Soret excitation of carbonmonoxy and oxy Hb and Mb photolyzes CO or Og, t n h i s t i d i n e f i f t h ligand. Although neither the Ni nor the Fe globins show evidence of photodissociation of the h i s t i d i n e ligand, the model compound data would, nevertheless, lead us to expect ligand ejection f o r Ni but not Fe. Time-resolved absorption (45-47) and transient Raman measurements (10,11) f o r 6-coordinate Ni-porphyrin complexes c l e a r l y predict that photodissociation of the f i f t h ligand w i l l occur, since g

n o t

e



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the transient species i s i d e n t i f i e d as the 4-coordinate form. On the other hand, the transient species i d e n t i f i e d i n the timeresolved absorption spectra of Fe-porphyrin 6-cojjdinate complexes i s a 5-coordinate form (63-65). For example, Fe -porphyrin(1methylimidazolejg complexes release only one of the imidazole ligands i n the excited state (63-65). The lack of photodissociation of the f i f t h ligand for the Fe-porphyrin complexes r e s u l t s from the nature of the transient excited state generated. The excited state has only one electron i n the d 2 o r b i t a l not two as f o r Ni; the state i s therefore l e s s d i s s o c i a t i v e . Thus, on the basis of the r e s u l t s f o r the model complexes release of the h i s t i d i n e ligand i s not predicted f o r the Fe globins (regardless of whether cage recombination occurs or not); ligand release i s expected f o r the N i reconstituted proteins but not o b s e r v e d . ^ Copper-reconstituted cytochrome c ( cyt-c) has also been investigated with transient absorption methods (49). No evidence of ghotoinduced e j e c t i o n of the f i f t h ligand i s observed i n either cyt-c or the model Cu-porphyrin 5-coordinate complexes (60-62). This i s consistent^with the l i k e l y transient state being a nondissociative π π or "> d 2_ 2 charge-transfer state. Instead, e x c i t a t i o n of 4-coordinaîe ïu-porphyrins i n coordinating solvents r e s u l t s i n weak binding of a f i f t h , but not a s i x t h , a x i a l ligand. E j e c t i o n of the ligand acquired i n the excited state occurs during or following deactivation through the charge-transfer state (62). The lack of photodissociation i n the Ni-reconstituted proteins argues f o r an impressive influence of the protein matrix on geminate recombination of the h i s t i d i n e ligand. The rebinding of the ligand i s even much f a s t e r than i n neat coordinating solvent where a ligand i s always available f o r uptake. The lack of s i g n i f i c a n t photodissociation may indicate the proteins maintain the h i s t i d i n e ' s o r i e n t a t i o n i n the "bound" p o s i t i o n even a f t e r photolysis has occurred. Holten and coworkers have also shown that 4-coordinate N i porphyrin species e x i s t i n g i n coordinating solvents are induced to pick up ligands by laser e x c i t a t i o n (45-47). This ligand-uptake process can also be understood i n terms of the d-electron configuration of the metal. Fog tê Ni-porphyrin molecules that have no a x i a l ligands, the (d 2 ) A^ state i s the groun^ state. Absorption of a photon by the tf -» π t r a n s i t i o n from the Aresults i n a d d t r a n s i t i o n moving one electron to the d r 2 o r b i t a l . The r e s u l t i n g half empty d 2 o r b i t a l then i s free accept charge from α-donor ligands. HjThe photoinduced uptake of ligands i s also observed i n the case of Hb. Unlike Mb, Hb i s a mixture of 4- and 5-coordinate forms. As shown i n Figure 4, e x c i t a t i o n of the 4-coordinate s i t e s in Hb ( e x c i t a t i o n i n the blue component of the Soret band) r e s u l t s i n a decrease i n the set of marker l i n e s corresponding to the 4coordinate form r e l a t i v e to the 5-coordinaê form. Since we observe the same photoinduced l i g a t i o n process i n Hb as observed f o r the model complexes i t i s reasonable to assume that the excited state l e v e l s of the 4-coordinate Ni-protoporphyrin system are not s i g n i f i c a n t l y altered by i t s protein environment. This further suggests that an a l t e r a t i o n of decay pathways i s not responsible f o r the d e l i g a t i o n behavior of the 5-coordinate species but rather rapid z

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geminate recombination. Most importantly, the rapid uptake of a f i f t h ligand on the 10-ns time scale suggests that the 4-coordinate Ni-porphyrin molecules are located i n the usual heme pocket where a f i f t h ligand i s r e a d i l y accessible.


Summary Resonance Raman s c a t t e r i n g provides a valuable method of determining the state of a x i a l l i g a t i o n i n nickel-reconstituted heme proteins and Ni-porphyrin complexes. A pattern of s h i f t s i n the Raman coresize and oxidation-state marker l i n e s can be used to monitor changes i n a x i a l coordination. The s h i f t s i n the core-size l i n e s (e.g. I/^Q) indicate an expansion of the core from about 1.96 Â f o r the 4coordinate Ni porphyrin to 2.04 Â f o r the 6-coordinate species, 22.03 A f o r the 5-coordinate species, and 2.01 Â f o r the unligated B- transient excited state. In 5-coordinate complexes the a x i a l ligand-Ni stretching mode has been i d e n t i f i e d . The mode can also be used to follow coordination changes. Five-coordinate Ni-porphyrin complexes have been observed, so f a r , only i n the Ni-reconstituted proteins. Transient Raman spectra i n the 10-ns time regime indicate changes i n l i g a t i o n state f o r the Ni-porphyrins i n aggreement with previous time-resolved absorption spectroscopic data. The 6coordinate Ni-porphyrin complexes that e x i s t i n coordinating solvents are induced to release ligands upon photoexcitation of the d i s s o c i a t i v e d 2_ 2 + d^2 state. E x c i t a t i o n of 4-coordinate species that e x i s t i n coordinating solvents induces ligand binding v i a the associative d 2 + d 2_ 2 t r a n s i t i o n . Although the same^transition i s expected f o r the 5-coordinate s i t e i n the Ni globins as f o r 6-coordinate models, ligand release i s not observed, presumably, due to rapid geminate recombination i n the protein. However, e x c i t a t i o n of the 4-coordinate s i t e of Ni hemoglobin does r e s u l t i n transient a c q u i s i t i o n of a f i f t h a x i a l ligand—most l i k e l y the proximal h i s t i d i n e . Other excited-state e f f e c t s besides coordination changes are observed i n the transient Raman spectra (10,11). Further analysis of the excited states and dynamics of Ni-porphyrin complexes and N i reconstituted heme proteins should benefit from Raman spectroscopy's inherently r i c h s t r u c t u r a l information content. Transient Raman methods are now being applied to other metalloporphyrins and metalloporphyrin-based systems. x

Z

Acknowledgments We thank Tomoko Yamamoto of the National I n s t i t u t e s of Health and Nai-Teng Yu of the Georgia I n s t i t u t e of Technology f o r h e l p f u l discussions. This work performed at Sandia National Laboratories and supported by the United States Department of Energy Contract DEAC04-76DP-00789 and the Gas Research I n s t i t u t e Contract 5082-2600767. Work at the University of New Mexico supported by the National I n s t i t u t e s of Health Grant GM33330, the donors of the Petroleum Research Fund administered by the American Chemical Society, and the C o t t r e l l research fund of Research Corporation. EWF was supported by the graduate fellowship program of the Associated Western U n i v e r s i t i e s .


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Axial Coordination in Nickel Porphyrins and Nickel-Reconstituted

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