2184
F. Adar, M.Gouterman, and S.Aronowitz
Fluorescence, Resonance Raman, and Radiationless Decay in Several Hemoproteins Fran Adar,* Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19 174
Martin Gouterman, and Sheldon Aronowltr Department of Chemistty, University of Washington, Seattle, Washington 98 195 (Received December 18, 1975)
Resonance Raman (RR) spectra are reported for five hemoproteins: (a) ferrocytochrome b5; (b) oxyhemoglobin; (c) ferricytochrome b5; (d) deoxyhemoglobin; and (e) methemoglobin. Compound a shows background fluorescence bands which give evidence for incomplete vibrational relaxation. Relative fluorescence yields for (a):palladium mesop0rphyrin:zinc mesoporphyrin are k2.5 X 102:104.Compounds (b) through (e) show no fluorescence but decreasing RR intensity. The presence of fluorescence in (a) and the decreasing RR intensity of (a) through (e) are attributed to decreasing electronic radiationless decay times, re,from 100 to 3 fs across the series (a) > (b) > (c) (d) > (e). The times re are estimated from the resolution of the visible absorption bands. Iterative extended Huckel calculations are used to rationalize re: Thus for (a) the visible band is lQ(r,a*) and a t lower energy the only state of the same spin is l(d,d); for (b) a t lower energy than lQ(a,r*) there is not only a possible l(d,d) state but singlets involving transitions on the 0 2 ligand and charge transfer singlets ring oxygen; for (c) the visible excited state is 2 Q ( r , ~ *and ) there are 2(d,d), 2(r,d), and 2T(r,r*)excited states at lower energy; similarly for (d) there are 5(d,d) and 5T(a,7r*) a t lower energy than the visible state 5Q(r,a*);finally (e) has both 6Q(r,a*) and 6(r,d) electronic states in the visible energy region with heavy vibronic mixing between them. The roles of photon coherence time, rp,and vibrational relaxation, rv,in determining the nature of the scattering process are explored.
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Introduction Recently there has been considerable interest in the resonance Raman spectra of many hemoprotein~l-~ as well as resonance Raman spectra of porphyrins coordinated to metals other than i r ~ n . In ~ - addition ~ there has been theoretical consideration of the relation between resonance Raman and resonance fluorescence.8-10 These phenomena reflect two aspects of the same physical process, whose differentiation depends on the relative lengths of the photon coherence time, r p ,and the lifetime of the resonant excited state, ri. The metalloporphyrins provide an ideal set of systems in which to study these phenomena because of the very strong dependence of excited state lifetime on the central metal.11-13 In this paper we shall report on the observed relationship of resonance Raman spectra (RR), resonance fluorescence (RF), and relaxed fluorescence (f) among several hemoproteins and metalloporphyrins with very short excited state lifetimes and relate these observations to the time durations of the various processes.
p2 main group metals such as Pb(II)] fluorescence yields are below and in some cases no fluorescence has been observed using conventional fluorimetric systems.11-19 However, with laser Raman apparatus detection of fluorescence yields can be pushed to lower levels, perhaps 10-6. Thus we can explore the emission properties of systems with excited state lifetimes in the range 60 ps to 60 fs. With such short lived systems it becomes important to distinguish the processes that contribute to ri, the lifetime of the initial excited state. There is of course radiative emission, with a lifetime given by eq 1.In addition there is the electronic state relaxation time, re. This is the process generally described as “radiationless decay”, which is presumed to be intramolecular and isoenergetic, from one electronic state to another.20-22 Also there is vibrational relaxation, occurring with time rv, in which the vibrational state decays through anharmonic coupling and through dissipation of its energy to the solvent environment. Thus the overall decay process is written Ti-1
Background For most metalloporphyrins the visible spectrum is assigned as a Q(a,a*) excited state.12 The natural radiative lifetime is determined from the absorption coefficient and for a wide variety of metals has a value14 rf
-
60 ns
(1)
Metalloporphyrins with closed atomic shells (e.g., do or dlO) show fluorescence yields, &, in the range 0.2 > & > 10-3.15,16 Excited electronic state lifetimes re in these cases can be determined from the relation +f = T e I T f
(2)
Hence they vary from about 12 ns to 60 ps. In the case of metalloporphyrins with partly filled shells [e.g., dn, 1 < n < 9, or The,Journal of Physical Chemistry, Vol. 80, No. 20, 1976
= rf-1
+ re-1 + r,-1
(3)
Finally, it has been noted that, in deciding which emission processes occur, the coherence time of the exciting photon, rp, must also be considered.8-10 The processes just defined have been related to the line width of various types of photons through the uncertainty relation Aib
-
(2rc)-’
(4)
where A; is the line width a t half maximum (cm-l) and r is the process decay time. For convenience these values have been listed in Table I. Equation 4 can be applied to the incident photon, which has a line width of -1 cm-l to get a value rp
-
5 ps
(5)
2185
Emission and Resonance Raman of Hemoproteins TABLE I: Line Width, Lifetime, and Fluorescence Yields for Metalloporphyrins A;, cm-l
dfU
7’1
530
10 fs
53
100 fs
3 fs 6 fs
1.7 x 10-7
15 fs 100 fs
1.7 X 1.7 x 10-5
1PS
5.3
le
1PS
Hb(II1) Hb(I1) HbOz cyt bdII)
-
~ ~ ( 3 K) 0 0 500 fsb
PtP
T ~15 ( K) 3 psC lp(laser) 5 psb N
5.3 x 10-1 5.3 x 10-2 5.3 x 10-3 5.3 x a
1.7 x 10-4 1.7 x 10-3 1.7 X 1.7 X 10-1
10 ps 100 ps 1ns 10 ns
12 ps
PdP
1.2 ns
ZnP
TAC = (27rc)-l; & = 1/60 ns. See text for basis of estimates. This value is poorest known; we only know 1.3 ps < T ,
< 12 ps. See
text. The line width of resonance Raman photons observed at room temperature is -10 cm-1, giving a vibrational relaxation time in the ground state of
-
500 fs (6) We shall be concerned with the nature of fluorescence as 7, becomes shorter than T ~ Over . the series of metal complexes zinc porphin (ZnP), palladium porphin (PdP), and platinum etioporphyrin (PtEtio) quantum yields, d f , have value 2 X 10-2,2 x 10-4, and 5 2 x 10-5.13J8h From Table I we see that 7, is 1.2 ns, 1 2 ps, and 51.2 ps, respectively; the uncertainty line widths would be 4.5 X lov3, 4.5 X lO-l, and -4.5 cm-l. The smallest line widths are observed in Shpol’skii matrices18 where a minimal line width at 4.2 K appears to be -3.2 cm-leZ3 Under these conditions it is necessary to write T,
= A?i 4-
(7)
&in
where APshp is the observed Shpol’skii line width, A?i the uncertainty broadening due to the excited state lifetime r i given by (3) and (4), and Aamm is a minimal line width due to other factors that are slightly temperature dependent. A minimal line width of 4 cm-l has been observed for ZnP and PdPlsc under similar low temperature conditions to those showing significantly broader Shpol’skii line width for PtP.18cSince conditions for these experiments are otherwise identical we assume that broader A&-,p observed for PtP is due to a shorter T,, in agreement with the estimate from q. The Aii,hp 4 cm-l observed for ZnP and PdP at 15 K implies T , > 1.2 ps in these low temperature matrices, a limit that probably holds for Pt complexes. Moreover, if T~ were this short one might expect the vibrational bands Q(1,O)of the visible Shpol’skii absorption spectrum to be broader than the Q(0,O) band. Since this is not observed,lscit is reasonable to presume that under these low temperature conditions T~ is rather longer than 1.2 ps. On the other hand, 7, is probably shorter than the electronic relaxation time of 12 ps. Thus we can say that 1 2 ps > T~ > 1.2 ps and estimate 1, 3 ps a t low temperatures. (An interesting point can be made concerning palladium porphyrin fluorescence. At room temperature, where T , > T , to one where 7, >
~ J R F= d 7 f
(2’) ~ , - l .The resonance
(8)
so that dRF/d)f
-
-
,
= Tv/Te
(9)
Equations 2’, 8, and 9 would, of course, have to be modified if several vibrational relaxations are needed to produce relaxed fluore~cence.~~b Equations 1-9 make it clear that, for metalloporphyrins, as observed fluorescence yield decreases below resonance fluorescence (RF) tends to dominate over relaxed fluorescence (f). The above kinetic arguments implicitly assume that the exciting photon is shorter lived than the molecular state, so that the interaction between the photon field and the molecule is complete before the molecule has a chance to rearrange itself. In fact for all cases reported here, since the emission studies were done at room temperature, eq 5 and 6 show that T ,
