Hydration and Solvent Accessibility of Zincsubstituted Cytochrome c

Solvent-mediated interactions often play key roles in protein folding reactions. Both folded and unfolded structures are associated with water molecules [67,68], and chemical denaturant activity is attributed to changes in water hydrogen bonding networks as well as direct solvation of peptide bonds and hydrophobic residues (Figure 4) [69,70]. Importantly, water and protein interactions are crucial for native structures [71], protein-protein recognition [72], and folding pathways [73-76]. Computational studies have suggested that the expulsion of water from the hydrophobic protein cavity could be an important step during folding. Molecular dynamics simulations reveal that rapidly-formed, collapsed structures trap a significant number of water molecules inside the protein [74]. Similarly, a theoretical model incorporating solvent effects found that the last step in protein folding involves 'squeezing out' water molecules from a partially hydrated polypeptide core [73]. While these computational studies have aimed to elucidate the role of water in protein structure and folding, very few experiments have probed the dynamics of solvation during a folding reaction.

Cofactor solvation in folded and unfolded protein states has been investigated using Zn(II)-substituted cyt c, which is structurally similar with comparable folding free energy to the native protein (Fe-cyt c) [43,77,78]. The key advantage of the Zn-protein is the availability of a long-lived (t ~ 15 ms), powerfully reducing (E° = -0.8 V vs. NHE) triplet excited state (*Zn-cyt c) [79,80]. Because the deactivation of *Zn-cyt c is strongly dependent on outer sphere solvation, high frequency O-H vibrations facilitate nonradiative decay. Significant deuterium isotope effects on decay rates have been reported because O-D stretches are less efficient acceptor modes [81,82]. Another channel for triplet state deactivation involves electron transfer to an external redox reagent, such as Ru(NH3)6+ [80], producing Zn-cyt c'+ and Ru(NH3)2+ (Figure 6). Since the degree to which there is an isotope effect or efficient triplet decay in the presence of quenchers directly reflects the extent of hydration and solvent accessibility of the Zn-porphyrin (Figure 4), each of these photophysical properties reports on the heme solvent environment of various conformational states of cyt c [43,45].

The cofactor in folded Zn-cyt c is partially hydrated, as indicated by the modest, yet reproducible, isotope effect kH2O/kD2O of 1.2 (Figure 7) [45]. This isotope effect is smaller than that observed for the fully exposed Zn-porphyrin in microperoxidase-8 (Zn-MP8), a heme octapeptide derived from enzymatic

Zn-cyt c

Zn-cyt c

Ru2+

Ru2+

Figure 6. Reaction scheme for bimolecular electron transfer quenching of triplet Zn-cytochrome c by Ru(NH3)3+.

NaPi

H2O

N\ D2O

H2O \\

d2o

5 M GuHCl

9 M Urea

h2o \\

d2o

1 I 11 III 1 1 1 11 II

1 1 II

tliij i i i 11 n

10-5 10-4 10-3 10-2 seconds

10-5 10-4 10-3 10-2 seconds

Figure 7. Transient absorption decay kinetics of triplet Zn-cyt c in protonated and deuterated solutions. Buffers were 20 mM phosphate buffer (top) with 5 M GuHCl (middle) or 9 M urea (bottom); solutions were pH = pD = 7.4.

cleavage of cyt c [83] (&h2o/&d2o ~ 1-5), and the triplet lifetime of the folded protein (t ~ 10ms) is much longer than that for Zn-MP8 (t ~ 300^s) [45]. The difference in lifetimes arises because the folded protein restricts solvent access to the cofactor (Figure 4). The observed isotope effect may be explained by the presence of water molecules in the Zn-porphyrin binding pocket of the folded protein [84] combined with partial cofactor exposure to the bulk solvent. The observation that numerous H2O molecules occupy conserved positions in a variety of cytochromes c indicates that these solvent molecules are critical for protein function [84].

Hydration of the cofactor in Zn-cyt c increases dramatically upon protein unfolding (Figure 4). The lifetime of the porphyrin triplet changes by nearly an order of magnitude, from ~10ms (folded) to ~ 1.3 ms (unfolded) (Figure 7) [43,45]. This change in lifetime cannot be attributed solely to the change in heme-solvent vibronic coupling; our studies with Zn-MP8 indicate that a change in cosolvent only affects triplet lifetimes by a factor of ~3 [45]. Instead, the decreased lifetime reflects enhanced Zn-porphyrin solvation, and this interpretation is further supported by the larger isotope effect for unfolded (k^O/^DO = 1-4) relative to folded (kH2O/kD2O = 1.2) protein [45]. Furthermore, by comparison to Zn-MP8 (k^O = 7000 s-1 in GuHCl), the triplet state decays nearly 10 times more slowly, suggesting that the cofactor is only partially exposed in the unfolded polypeptide (kH O = 810 s-1 in GuHCl) [45].

The presence of this shielding supports the notion that unfolded proteins are not random polymers, but instead may adopt partial structure even under fully denaturing conditions [66,85-88]. An unfolded structure may feature hydrophobic clustering [86], native-like topology [85], or in the case of heme proteins, well-defined heme ligation geometry [87]. The existence of such partially structured unfolded states with locally buried regions also can be inferred from the results of bimolecular ET quenching experiments with Ru(NH3)6+ (Figure 6). The reported quenching rate constant measured under native conditions is 1.4 x 107M-1 s-1 [80]. Under denaturing conditions ([GuHCl] > 3M), the quenching reaction is 100 times faster (1.4 x 109M-1 s-1, [GuHCl] = 3.5M) than that measured in the absence of GuHCl [43]. The quenching rate is very high, owing to the greater accessibility of the Zn-porphyrin in the unfolded protein. The *Zn-cyt c ET kinetics are generally consistent with a two-state unfolding process; and the unfolding isotherm generated from ET kinetics exhibits a transition midpoint at 2.8(1) M GuHCl, in good agreement with those obtained from far-UV CD and heme absorption measurements.

A molecular description of the mechanism of chemical denaturation is a goal of much current research [70,89-91]. For Zn-cyt c, the free energies of unfolding are comparable in urea (AG° = -31 kJ/mol) and GuHCl (AG° = -35kJ/mol). In high urea and GuHCl concentrations Zn-cyt c exhibits similar triplet lifetimes (800 s-1 in GuHCl; 700 s-1 in urea) and isotope effects (kH O/kD O = 1.4

in GuHCl and urea) [45], suggesting nearly identical solvent exposure of the Zn-porphyrin.

In contrast to fully denatured protein, the nature of the compact species depends on denaturant. Bimolecular quenching of the triplet Zn-porphyrin in the compact state in urea is times slower than in GuHCl (5.9 x 107M-1s-1 in 2.7 M GuHCl; 8.6 x 106 M-1 s-1 in 6.7 M urea), and this difference likely reflects greater protection of the porphyrin group from the aqueous urea solvent [45] as well as electrostatic effects, since it has been shown that triplet Zn-cyt c is sensitive to ionic strength and quencher charge [92,93]. However, the bimolecular quenching rates observed here are similar to those reported for folded and molten globule states of Zn-cyt c [93], supporting our interpretation that the rate differences are attributable to variations in the equilibrium GuHCl- and urea-induced compact structures. It is possible that these partially unfolded species are similar to the intermediates associated with the burst phase of cyt c folding [31,43,48,77,94]. The compact structure likely represents this partially unfolded intermediate as well, and the finding that the structure of this species is denaturant-dependent supports the notion of nonnative heme environments for this compact, partially unfolded species.

The observation of more collapsed nonnative structures in urea may reflect the relative strengths of these two widely used denaturants. There is general consensus that chemical denaturants act through a combination of direct binding to the peptide backbone and side chains, and by altering the hydrogen bonding network of water in a structure-making or structure-breaking manner, thereby diminishing the hydrophobic effect [69,70]. It is well established that the transfer free energy for the peptide group from water to aqueous solutions of urea or GuHCl is favorable, with GuHCl being a better solvent for the peptide backbone than urea [95]. In contrast, side chains show wide variability in transfer free energy to denaturant solutions; however, for a given denaturant concentration, GuHCl is more effective in its ability to solvate both hydropho-bic and hydrophilic residues [96,97]. Studies on charge effects suggest, not surprisingly, that denaturation by urea is more sensitive to protein charge and ionic strength than GuHCl [90,91,98,99]. Collectively, these reports indicate that GuHCl is more effective than urea in its ability to disrupt and solvate hydropho-bic pockets of folded proteins, in accord with the finding that the Zn-porphyrin in the compact state of Zn-cyt c is more exposed to solvent in GuHCl than in urea.

It is clear that Zn-porphyrin triplet lifetimes are sensitive indicators of heme-pocket hydration. The finding that folded and unfolded species have different isotope effects and bimolecular quenching rates indicates that changes in hydration can be monitored during a folding reaction (Figure 4). Important issues such as the time scales for water expulsion, the nature of dehydrated and hydrated intermediates, and even the effect of denaturant on folding pathways can be addressed by means of triplet lifetime measurements.

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