Chapter 3
Functional Changes in the M121E Mutant of Azurin

Marcus-type analysis of ET rates

The Marcus theory of electron transfer has proven to be remarkably successful in explaining the important factors that determine ET rates in a variety of situations, from ET between small inorganic complexes in solution to ET within large biological complexes like the photosynthetic reaction center (Marcus and Sutin 1985). The semiclassical formulation of the original rate equation is an appropriate level of analysis for most biological systems:

kET = 2 exp-

In this formulation the solvent is treated classically and the electronic coupling is treated as a quantum phenomenon. Independent measurement of all of the factors affecting the electron transfer rate, T, DGŚ, HAB, and l, is difficult. However, inferences about less directly accessible variables such as HAB or l can be made by spectroscopic measurements or by examination of ET rates for a series of DA systems where either of the more manipulable variables, T or DGŚ, is systematically varied.

In this chapter I will discuss the electron transfer rates reported in the previous chapter within the context of the semiclassical Marcus equation in an effort to determine how the substitution of a glutamic acid residue for the normal methionine ligand affects the functioning of the blue copper site.

Driving force

The free energy change for any chemical reaction is the difference in the energy of the products and the reactants. For electron transfer reactions this can be broken into the work it takes to bring the donor and acceptor together and the difference between the reduction potentials of the acceptor and donor. Since we are concerned here only with intramolecular electron transfer reactions, we can neglect the work term. For the flash-quench experiment and the back electron transfer in the photoinduced experiment, the Ru(bpy)2ImHis3+ label is the electron acceptor. The reduction potential for the 3+/2+ couple for a Ru(bpy)2Im2 model complex has been measured to be 0.98 eV (Casimiro et al. 1993). The reduction potential of the complex bound to a protein changes very little. The reduction potential of Ru(bpy)2Im bound to H33 of cyt c has been directly measured to be 1.07 eV (Mines et al. 1996). The reduction potential of the complex bound to H83 of azurin is 1.082 V (Di Bilio et al. 1997). For the forward reaction in our photoinduced electron transfer scheme, the electron donor is the excited state of Ru(bpy)2ImHis2+*. This complex is a good electron donor and its reduction potential, estimated from difference between the energy of the excited state emission of the Ru2+ complex and the reduction potential of Ru(bpy)2Im23+/2+, is calculated to be -1.03 eV (Mines et al. 1996). (See figure 1.)

Figure 3.1 Reduction potentials of various Ru(bpy)3 species (Roundhill 1994). From data summarized in (Mines et al. 1996), the analogous potentials for the 3+/2+/2+* triangle for Ru(bpy)2ImHis33 cytochrome c would be: E00 = 2.1 V and EŚ for Ru3+/2+ = 1.07 V, EŚ for Ru2+/1+ = -1.03 V.

The reduction potentials of azurin and mutant forms thereof can be measured by spectroelectrochemistry (Taniguchi et al. 1980). The reduction potentials of type 1 copper proteins are all higher than the 115-150 mV potential of most inorganic Cu2+/1+ complexes (Canters and Gilardi 1993). This, and more particularly, the large variation in reduction potentials of blue Cu proteins with very similar spectroscopic properties (184 mV for stellacyanin to 780 mV for fungal laccase (Taniguchi et al. 1982)) has been the subject of much discussion in the bioinorganic literature (Gray and Malmstr¸m 1983; St. Clair et al. 1992; Pascher et al. 1993). In most blue copper proteins, the Cu ion is buried under the surface of the protein in a fairly rigid, hydrophobic site. The enthalpy of reduction for the blue copper proteins is favorable (DHŚ = -16.6 kcal/mol for WT P. aeruginosa azurin), probably because the reduced protein has an electrically neutral Cu site buried in the hydrophobic interior (Cu1+ neutralized by the cysteine thiolate). The reaction entropies for reduction of blue copper proteins, however, are all negative (unfavorable), indicating an increase in order around the reduced copper protein (Taniguchi et al. 1982).

The reduction potential of WT azurin has also been observed to have a moderate pH dependence. Between pH 8.0 and 5.0, the potential increases from 292 mV to 349 mV (St. Clair et al. 1992). Formerly, this increase had been attributed to structural rearrangements that accompany the protonation of H35. Crystal structures of the oxidized protein at pH 5.5 and 9.0 show that protonation of H35 leads to formation of a strong hydrogen bond to the P36 carbonyl oxygen causing a change in the conformation of the peptide bond between P36 and G37 (Nar et al. 1991). This peptide bond flip causes changes in the adjoining loop regions but only very small changes in copper center, thus there is no pH-dependence in the spectroscopic properties of the wild type protein. The 60 mV increase in the WT reduction potential at low pH is consistent with an additional positive charge in the vicinity of the Cu site favoring the Cu1+ oxidation state. However, the H35K mutant shows very little change in reduction potential compared to wild type and the pH dependence of the potential remains nearly the same, effectively ruling out H35 protonation as the source of the pH dependence of the WT azurin reduction potential (Pascher et al. 1993).

Changes in the ligand residues of P. aeruginosa azurin have been made using site directed mutagenesis. Surprisingly, changes can be made in all four side-chain ligands while still retaining the ability to bind copper. Although the blue color of the site is abolished, the C112D mutant binds copper and its reduction is reversible. The site's reduction potential is estimated from redox titration with cytochrome c551 to be ~180 mV (Mizogouchi 1996). Glycine substitutions have been made for both histidine ligands and the sites can be converted to spectroscopically normal type 1 centers by adding imidazole ligands. However reduction of H117G, H46G, and all of their substituted derivatives is irreversible. This inability to reoxidize the H-X-G mutants is not due to chemical modification of the protein since one can remove the Cu1+ from H46G and reconstitute the protein with Cu2+. It may be due to an increased reduction potential because a three-coordinate site stabilizes the Cu1+ form relative to the Cu2+ form (van Pouderoyen et al. 1996). Reduction of a His46 mutant containing a covalently attached ligand (His46Asp) is reversible, although its potential is somewhat lower than WT, in line with the increased polarity at the Cu site (Germanas et al. 1993).

Proteins containing all 20 natural amino acids at position 121 have been isolated (Chang et al. 1991; Karlsson et al. 1991). Many show very little change from the WT reduction potential. The changes observed correlate with the polarity of the site. Deleting the entire last b-strand in the M121End mutant increases the solvent accessibility of the site and drops the reduction potential to 205 mV. When the uncharged but polar methionine is changed to a non-polar leucine residue, the reduction potential at pH 7.0 is increased by 138 mV to 448 mV (Pascher et al. 1993). The reduction potential of one of the M121 mutants, M121E, shows an interesting pH dependence. At low pH it is rather similar to WT (370 mV at pH 4.0) but decreases dramatically with increasing pH, going to 184 mV at pH 8.0. This, along with the changes in the optical and EPR spectra seen with this mutant, have been taken as evidence of changes in Cu coordination. When the axial glutamic acid ligand is deprotonated, it is presumed to bind the Cu, altering the spectroscopy of the site and decreasing the reduction potential by favoring the Cu2+ state (Karlsson et al. 1997).

Taking the difference between their reduction potentials, one can calculate the driving force (-DGŚ) for electron transfer from the reduced M121E Cu center to the oxidized Ru label: 0.71 eV at low pH, 0.90 eV at high pH. The driving force for ET from the WT center (at pH 7.0) is 0.76 eV. Despite these 50-140 mV changes in driving force, calculations predict very little change in the electron transfer rates. (See figure 2.) If there are no changes to other ET parameters, one would expect the rates from M121E to H83 to be essentially the same as those from the wild type center. For the more closely coupled acceptor at H122, one would expect to see a small decrease in the M121E rates because their driving forces are not quite as close to the measured l (0.80 eV (Di Bilio et al. 1997)) as the WT driving force.

Figure 3.2 Calculated and observed electron transfer rates to ruthenium labels at positions 83 and 122 in azurin.

 

H83

H122

 

calculated

observed

calculated

observed

WT

 

1.2 x 106 (a)

 

7.1 x 106 (b)

Low pH

1.1 x 106

4.1 x 105

6.6 x 106

1.9 x 106

High pH

1.1 x 106

5.0 x 105

6.5 x 106

1.3 x 106


(a) (Di Bilio et al. 1997)
(b) (Langen et al. 1995)

Our results are not consistent with these expectations. The M121E ET rates are much lower than predicted rates. Clearly the naive assumption that only the driving force changes is incorrect. This implies that either the electronic coupling or the reorganization energy is different in the M121E azurin mutant.

Electronic coupling

As discussed in the introduction, the original motivation for this project was to determine if the spectroscopic changes seen in the M121E mutant at high pH would translate into increased electronic coupling between the copper center and labels placed on the 121-126 b-strand. An X-ray structure of the M121E mutant in the low pH form shows a closer interaction between the Cu2+ ion and the O of the glutamic acid ligand: a Cu-O bond distance of 2.21 ╩, as compared to 3.15 ╩ for the Cu-S interaction in the WT center (Karlsson et al. 1997). (See figure 3.) But at low pH the M121E site is a fairly typical type I blue site - both spectroscopically and in terms of its reduction potential. Thus, one might expect the electron transfer rates from the M121E center to be about the same as those from the WT center.

Figure 3.3 Oxidized copper site of the M121E mutant of P. aeruginosa azurin (Karlsson et al. 1997). The copper ion is ligated by four out of the five usual ligands, C112 (to the left and behind the Cu ion in this view), H117 (left front), H46 (right), and the carbonyl oxygen of G45 (below and to the right). The introduced glutamic acid side chain (top center) interacts with the Cu2+ ion via one of its oxygens. The resonance Raman spectrum of the crystal shows enhancements characteristic of the blue, low-pH form of the protein, indicating that this structure shows the position of the protonated glutamic acid. The heavy atoms are colored according to the standard CPK scheme: carbon: gray, nitrogen: blue, oxygen: red, sulfur: yellow. A similar view of the wild type center is shown in figure 1.6.

At high pH, the deprotonated glutamic acid side chain interacts more strongly with the Cu ion. Evidence for this increased interaction includes an additional coordinating oxygen seen in the EXAFS (Strange et al. 1996), shifts to higher energies of the LMCT bands in the M121E Ni2+, Co2+, and Cu2+ derivatives (Di Bilio et al. 1992), and increased rhombicity in the EPR spectrum at high pH (Karlsson et al. 1997). This increased metal-ligand interaction upon deprotonation would be expected to increase the electronic coupling (HAB) into the Cu site, leading to higher electron transfer rates at high pH.

The observed ET rates for the M121E mutant do not conform to these initial expectations about changes in electronic coupling. All of the rates are lower than expected and while the M121E/H83 ET rates increase slightly with increasing pH, the M121E/K122H ET rate decreases at high pH - in direct contradiction to expectation if the electronic coupling through the new glutamic acid ligand increases.

Recent work has shown that changes in ligand residues that have very small effects on the spectroscopy of a blue Cu site can have dramatic effects on the electron transfer properties of the mutant (Regan et al. 1998). Changes in the paramagnetic NMR shifts of ligand protons indicate that the H46D mutation induces an increase in the interaction of the Cu with the axial methionine, accompanied by a decrease in its interaction with the CysS (Vila et al. 1997). This is seen as a subtle change in the spectroscopy of the site; the H46D azurin has a ~600 nm band that is slightly blue shifted and has a lower extinction coefficient than the WT LMCT. However, this small decrease in coupling between the Cu and its cysteine ligand leads to a dramatic decrease in the ET rate from the mutant site (a 36 fold drop from 1.2 x 106 to 3.2 x 104 s-1).

The low pH spectrum of M121E shows an analogous blue shift and decrease in extinction coefficient of the LMCT band (Karlsson et al. 1997) and the ET rates are also significantly lower than wild type but they are only 2-6 fold lower, as opposed to 36 fold lower in the H46D mutant. And, more importantly, the decrease in rate does not correlate with the extent of change in the site's LMCT band. The low pH rate to H83 decreases more than the high pH rate but the converse is true for ET to labels at H122. So, while the M121E substitution may have some effect on the electronic couplings in the Cu site, either by changing the coupling through the axial ligand into the b-strand, or indirectly through its effects on the Cu-Cys bond, this does not seem to supply an internally consistent explanation for the rate changes seen.

Reorganization energy

All the rates reported here were taken at 298 K, so the other variable in the Marcus equation is the reorganization energy. According to the Marcus cross relation, the reorganization energy of a reaction is the average of the reorganization energies of the two components:

l12 =

These reorganization energies can be broken down further into inner and outer sphere components:

l = li + lo

The inner sphere reorganization component is the energy required to alter bond distances and bond angles that change with the change in oxidation state. The outer sphere reorganization is the energy required for reorientation of the solvent around the changed complexes.

In blue copper proteins, the electron transfer reorganization energies are kept low by the small changes in ligand coordination (low li) and minimal solvent reorganization (small lo) because the metal centers are separated from bulk solvent, buried beneath the hydrophobic patch at the 'northern end of the molecule. X-ray structures of oxidized and reduced WT Alcaligenes denitrificans azurin show small increases (0.05 - 0.1 ╩) in all Cu-ligand bond lengths upon reduction, commensurate with the increase in radius of the Cu1+ ion; the copper atom is not displaced from the HisHisCys plane (Shepard et al. 1990). X-ray data are not available for reduced P. aeruginosa azurin but EXAFS data show similarly small changes in bond distances - with the possible exception of an increase in the MetS-Cu distance in the oxidized protein (Murphy et al. 1993). In addition, Loppnow and coworkers have studied the inner sphere reorganization energy of the WT azurin charge transfer interaction by analyzing the absolute cross section of the resonance Raman spectra (Webb et al. 1997). Using this technique they were able to estimate the relative contributions to the relaxation of the excited CT state of population decay (relaxation into lower lying CT and LF states of the protein), specific vibrational modes, and solvent-like dissipation of energy. Adding up the mode-specific reorganization energies (l for each RR band), they obtain 0.26 eV for the reorganization energy due to specific vibrational displacements. From the homogeneous line widths they further estimate that the rest of the protein provides 0.12 eV of 'solvent-like' reorganization energy. Their estimate of 0.38 eV for the total inner sphere reorganization during charge transfer further supports a low value of linner for the electron transfer reactions of WT azurin.

Recently, Di Bilio measured the reorganization energy of the azurin WT Cu center (Di Bilio et al. 1997). By analyzing the driving force and temperature dependences of ET rates for a series of azurins modified at H83 with Ru and Os compounds, he obtained a l for the ET between the Cu center and the Ru(bpy)2Im label of 0.80 eV. Taking a reorganization energy of 0.78 eV for the Ru(bpy)2Im label (estimated from ET studies with labeled cyt c), the reorganization energy of the blue copper center is 0.82 eV. Farver and Pecht have estimated the reorganization energy of ET from the disulfide bond at the 'southern' end of azurin to the Cu center to be 1.03 eV (Farver and Pecht 1997). This does not give us an independent measure of the reorganization energy of the Cu site alone because reorganization energy of the disulfide bond is not known. However, given the substantial bond length change for the disulfide radical, the S-S reorganization energy would be expected to be substantial, so 0.82 eV for the Cu center is in reasonable agreement with their expectations.

While direct determination of reorganization energies requires either a driving force or temperature dependence study, estimates for the reorganization energies of the M121E site can be made by assuming that the electronic coupling remains constant and calculating the reorganization rates from the ratio of two electron transfer rates.

= exp-

Calculations comparing WT ET data from the 122 and 83 sites with M121E data give consistent reaction reorganization energies for each pH, 1.08 eV at low pH and 1.27 eV at high pH. Using the reorganization energy for Ru(bpy)2Im, l11 = 0.78 eV, and the Marcus cross relation, l12 = (l11 + l22)/2, calculations predict reorganization energies of 1.38 eV at low pH and 1.76 eV at high pH for the M121E Cu site. The assumption that there is no change in electronic coupling through the cysteine S-Cu bond is naive so these calculated reorganization energies represent an upper limit for the changes in the M121E Cu site.

Increased reorganization energies make sense if one examines the structure of the Cu site. The reorganization energy of the WT Cu center is low (l22 = 0.82 eV) because the Cu ion in either oxidation state is protected from the surrounding aqueous medium. The crystal structure of the WT center shows that the Met ligand blocks access of the solvent to the Cu ion but is only weakly coordinated (Cu-S distance 3.15 ╩ (Nar et al. 1991)). A crystal structure of the M121E mutant shows that, even in the protonated form, the glutamic acid ligand is well coordinated (Cu-O bond distance 2.2 ╩ (Karlsson et al. 1997)). In addition the M121E site is more polar, even when the glutamic acid residue is protonated. This leads to a more variable interaction with the Cu ion in its two oxidation states and a higher reorganization energy for the electron transfer reaction. At high pH, the glutamic acid residue is deprotonated becoming not only polar but negatively charged. This negative charge would be predicted to increase both the degree of inner sphere reorganization and the reorganization of the solvent around the site during oxidation.

Corroboration for increased mobility of the Cu site in the M121E mutant comes from perturbed angular correlation (PAC) studies (Danielsen et al. 1995). Data from several cadmium-substituted M121 azurin mutants show that the mutant Cu sites are less rigid than the WT site. Several of the mutants (Ala, Leu, and Glu) required two different nuclear quadrapole interactions (NQI) to model the site effectively. With the Ala and Leu mutants, one NQI is three coordinate (Cys, His, His) like the WT center while the other could best be described as four coordinate with an additional water molecule in the site. The data from the M121E mutant could not be modeled starting from the WT structure. However, one of the NQI's is modeled well starting from the crystal structure of the M121E mutant where the Cu is seen to be coordinated by C112, H46, H117, and one of the E121 oxygens. The other NQI may represent the site with E121 coordinated in a bidentate fashion. A further indication of decreased rigidity in the M121 mutant sites is an increase in the linewidths of the PAC spectra.

Conclusions

In summary, despite clear spectroscopic indications of increased ligand-metal interaction in the high pH form of the M121E mutant of azurin, this does not translate into increased electronic coupling to donors placed on b-strand leading away from that ligand. Our efforts to increase electron transfer rates through that section of the protein by the electronic coupling between the donor (the azurin Cu atom) and the bridge (the intervening protein) were thwarted by the simultaneous alteration of the nuclear factors, in particular a dramatic increase in the reorganization energy of the mutant Cu site. This gives a possible explanation for the puzzle of the almost absolute conservation of the methionine ligand in blue copper proteins despite the near normal stability and spectroscopy of substitutions at that ligand position. Methionine is a fairly large, polarizable but not very polar group; these properties help it exclude water from the Cu site while interacting minimally with the Cu ion. This is important in helping to tune the reduction potential of the site and, more importantly, in minimizing the reorganization energy during electron transfers.

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