An outer-sphere hydrogen-bond network constrains copper coordination in blue proteins

Journal of Inorganic Biochemistry 88 (2002) 375–380 www.elsevier.com / locate / jinorgbio

Focused Review

An outer-sphere hydrogen-bond network constrains copper coordination in blue proteins 1

Michael C. Machczynski , Harry B. Gray*, John H. Richards* Beckman Institute, MC 139 -74, California Institute of Technology, Pasadena, CA 91125 -7400, USA Received 19 June 2001; received in revised form 1 January 2002; accepted 8 February 2002

Abstract In azurins and other blue copper proteins with relatively low reduction potentials (E 0 [Cu II / Cu I ],400 mV vs. normal hydrogen electrode), the folded polypeptide framework constrains both copper(II) and copper(I) in such a way as to tune the reduction potentials to values that differ greatly from those for most copper complexes. Largely conserved networks of hydrogen bonds organize and lock the rest of the folded protein structure to a loop that contains three of the ligands to copper. Changes in hydrogen bonds that allow copper(I) to revert more closely to its preferred geometry [relative to the copper(II) geometry] accordingly lead to an increase in E 0 . This paper reports mutations in the ligand loop of amicyanin from P. denitrificans that relax the constraints on ligation for copper(I) and significantly raise E 0 for these mutants (for example 41564 mV) relative to that of the native amicyanin (26564 mV). These mutations also shift the pKa of a ligand histidine to below 5 relative to 7.0 in the wild type.  2002 Elsevier Science Inc. All rights reserved. Keywords: Reduction potential; Copper; Blue copper proteins; Azurin; Amicyanin

1. Introduction In blue copper proteins, the surrounding polypeptide orders the geometry of the copper ligation so as to tune the reduction potential E 0 , and lower the reorganization energy l, to facilitate the transfer of electrons to satisfy the metabolic needs of many organisms [1–5]. To generate reduction potentials that are significantly higher than that for aqueous cupric ion, these proteins orient the ligand environment for the Cu(II) form to be close to that of Cu(I) as the crystal structures of many blue copper proteins, beginning with that of poplar plastocyanin in 1978 [6], abundantly document. Importantly, the polypeptide fold itself determines the ligand geometry for copper. Thus, plastocyanin and apoplastocyanin [7] as well as azurin and apoazurin [8] have the same three-dimensional structures; binding of copper does not significantly alter the orientation of the copper ligands. Because of this central role of the structure of the

*Corresponding authors. Fax: 11-626-4494-159. E-mail addresses: [email protected] (H.B. Gray), [email protected] (J.H. Richards). 1 Current address: Metalloprotein Group, Gorlaeus Laboratories, Einsteinweg 55, Leiden University, 2333 CC Leiden, The Netherlands.

polypeptide chain in regulating the ligand geometry around copper with the resultant biochemically crucial influence on the electron transfer properties of these proteins, the question of whether the protein more constrains the Cu(II) or Cu(I) redox state has attracted considerable attention [1–5,9–15] and is the central focus of this paper. The crystal structures of plastocyanin at low pH showed that the C-terminal histidine ligand in the reduced form of the protein becomes protonated and detached from the copper (which is now tridentate) [16]. This observation of a redox-coupled conformation change hinted that copper(I) might, in fact, be more constrained than copper(II) in folded blue copper proteins. The structures of pseudoazurin depend similarly on the redox state of the copper [17] as do those of amicyanin [18], stellacyanin [19] and rusticyanin [20].

2. Results Changing the ligand loop, particularly in ways that might free it somewhat from its normally tight association with the rest of the protein, provides an avenue for examining the effect that the resultant relaxation of the ligand environment may have on the redox properties of the copper and other aspects of the behavior of these

0162-0134 / 02 / $ – see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 02 )00364-1

M.C. Machczynski et al. / Journal of Inorganic Biochemistry 88 (2002) 375 – 380

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proteins. In this work, these changes were introduced by creating two mutants of proline residue 94 in Paracoccus denitrificans amicyanin, specifically P94A and P94F. Both of these mutants have considerably higher reduction potentials [by 115 mV (P94A) and 150 mV (P94F)] than the parent amicyanin (see Table 1). Comparing the temperature dependences of the native and P94F mutant indicates that reduction leads to an increase in structural flexibility of the Cu(I) form of the mutant relative to the wild type. Thus, while the enthalpies of reduction are virtually identical (24162 kJ / mol for both) the entropies of reduction differ markedly (253.368 J / mol K for the wild type vs. 28.665 J / mol K for the P94F mutant). Notably, the visible absorption maximum shifts from 596 (wild type) to 608 nm (P94F mutant) and the electron paramagnetic resonance (EPR) parallel hyperfine splitting slightly increases (from 74310 24 to 95310 24 cm 21 ), which indicates diminished copper–thiolate covalency. A further significant change in the P94F mutant is a lowering of the pKa for the C-terminal histidine ligand from 7.0 (wild type) to less than 5.0.

Table 1 Reduction potentials of wild type and mutant blue copper proteins Protein

Organism e

E 0 [Cu(II / I)] (mV vs. NHE)

Conditions

Azurin wild type

P. aeruginosa

310 a

pH 7.0, 0.1 M NaPi

Azurin 1 M GdnHCl

P. aeruginosa

420 a

pH 7.0, 0.1 M NaPi

Azurin wild type

A. denitrificans

286 b

pH 7.0, 0.1 M NaPi

Azurin N47L mutant

A. denitrificans

396 b

pH 7.0, 0.1 M NaPi

Pseudoazurin wild type

A. faecalis

270 c

pH 7.0, 0.1 M KPi

Pseudoazurin P80A mutant

A. faecalis

409 c

pH 7.0, 0.1 M KPi

Pseudoazurin P80I mutant

A. faecalis

450 c

pH 7.0, 0.1 M KPi

Amicyanin wild type

P. denitrificans

265 d

pH 8.0, 0.1 M NaPi

Amicyanin P94A mutant

P. denitrificans

380 d

pH 8.0, 0.1 M NaPi

Amicyanin P94F mutant

P. denitrificans

415 d

pH 8.0, 0.1 M NaPi

a

Ref. [33]. Ref. [38]. c Ref. [40]. d Ref. [36]. e P. aeruginosa5Pseudomonas aeruginosa; A. denitrifocans5 Alcaligenes denitrificans; A. faecalis5Alcaligenes faecalis; P. denitrificans5Paracoccus denitrificans. b

3. Discussion These results further document the role of structural constraints in the Cu(II) and Cu(I) forms of these proteins and provide evidence for a greater relative constraint for the Cu(I) than for the Cu(II) protein. Further, they accord with other evidence that supports a similar view. Such evidence includes the observation that the substitution H117G in azurin creates a binding site that becomes occupied by solvent or exogenous ligand [21]. Strikingly, spin echo modulation (ESEEM) measurements show that exogenous imidazole binds Cu(II) in the same orientation as the imidazole group of the histidine side chain in the wild type protein [22]. Reduction of Cu(II) to Cu(I) in this mutant causes ejection of the imidazole from the protein, leaving the metal tricoordinate like that in the Cu(I) plastocyanin and suggests that Cu(I) is more constrained by the protein backbone than Cu(II). Density functional theory (DFT) also sheds light on this question. Cu(II)(His) 2 (Cys)(Met) structures are not strained [3,23,24] and calculations with an improved basis set show that an imidzaole ligand would dissociate from an isolated Cu(I) complex of this type. Placing such a metal site in a protein-like medium, in particular by including hydrogen bonds to the cysteine thiolate, increased the accuracy of the calculations which were then able to obtain close agreement with the known copper-ligand bond lengths and angles [24]. Further along these lines, only the Cu(II) states of synthetic analogues of the active sites of copper proteins can be characterized. Employing bulky ligands, Kitajima’s group prepared Cu(II) complexes that exhibit spectroscopic properties very similar to those of the blue copper proteins themselves [25–27]. However, the redox properties of these spectroscopically satisfactory models differ from those of the proteins significantly; indeed, the reduction potential, measured in methylene chloride, is negative (2540 mV vs. a silver wire). More recently, Holland and Tolman have synthesized copper complexes that utilize the same ligand set as the proteins [28,29]. These models well reproduce the bond lengths when they lack an axial methionine and the Cu(II) form favors a slightly out-of-plane geometry. Addition of an axial thioether causes a significant shift to a squashed tetrahedron similar to that in mutants of the binuclear CuA site of cytochrome c oxidase [30]. Again, the reduction potentials measured in aprotic solvents fall significantly below those of the proteins themselves, indicating that Cu(I) is greatly disfavored relative to Cu(II) in this coordination geometry. Hydrogen bonding in the vicinity of the copper likely plays important roles in determining the relative constraints of Cu(II) and Cu(I). This in turn influences the reduction potential as well as the relative stability of the Cu(II) and Cu(I) forms of the protein [31,32]. Thus, the

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midpoint of the unfolding curve of Cu(II) azurin is at 3.9 M GdnCl whereas the midpoint for Cu(I) azurin is 2.6 M GdnCl [(that for the Zn(II) derivative is 2.75 M GdnCl]. Further, electrochemical experiments on azurin in denaturing solvents reveal the existence of a high potential intermediate (420 mV vs. NHE) with an unfolding midpoint at 1 M GdnCl [33]. The characteristic visible

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absorption shifts less than 2 nm in the spectrum of this species, indicating no significant perturbation to the structure of the Cu(II) inner sphere. The 110 mV upshift in potential (Table 1) from that of the native protein was attributed to the breaking of hydrogen bonds in the active site region of the coordination geometry of the Cu(I) (Fig. 1).

Fig. 1. Active sites of P. denitrificans amicyanin and A. denitrificans azurin. The green ribbon represents the backbone of the amicyanin ligand loop, consisting of only seven amino acids. The stick model is based on the crystal structure of azurin, with hydrogen bonds appearing as dashed green cylinders. The ligand loop of azurin is 11 amino acids long. Hydrogen bonds stabilize the metal site and adapt for differences in loop lengths, demonstrated by the similarities in the figure. The numbering for azurin residues is shown. (A) A hydrogen bond from a sidechain carboxyl group (Asn10) to the N-terminal histidine ligand (His46), fixing its orientation. The copper is directly above the imidazole ring and is eclipsed by other atoms and the ribbon. (B) The ‘‘zipper’’ region contains three nearly parallel hydrogen bonds that connect the ligand loop to the rest of the protein. The lower bond connects the backbone amide (Asn47) to the cysteine thiolate (Cys112, in yellow), while the other two bonds restrain a serine (Ser113) in the loop. Severing these bonds results in copper loss and / or increased reduction potential. Slightly to the left of the zipper, visible through the serine, is a second hydrogen bond to the thiolate; this contact is found only in azurins and similar proteins with large loops. (C) This triangle consists of bonds between the backbone amides and carbonyls of the Cys, His, and Met ligands in many loops (azurin uses the amide after the His residue). The location and orientation of these bonds, coincident with both the amicyanin and azurin ligand backbones, suggest a crucial structural role. (A), (B) and (C) are conserved among blue copper proteins, and occupy the same spaces in analogous crystal structures (amicyanin set omitted for clarity). (D) Elements responsible for strain in the large azurin loop include two intraloop (His117–Phe114 and Met120–His117) hydrogen bonds and a proline (Pro115).

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In A. denitrificans azurin, 11 of the 16 hydrogen bonds between residues distant from each other in amino acid sequence occur in the loop which contains the cysteine and two histidine ligands (the ligand loop) [34]. Three residues of this loop and a conserved Asn in the N-terminal His strand serve as a zipper between the metal binding ligand and the main beta barrel structure of the protein [35]. Severing all three of these hydrogen bonds results in loss of the copper [36,37]. A N47L mutation in A. denitrificans azurin removes two hydrogen bonds to the Ser in this zipper but retains the hydrogen bond to the cysteine thiolate. In the N47L mutant the reduction potential shifts to 396 mV vs. NHE from 286 mV vs. NHE in the wild type [38]. This increase in potential likely reflects a local unfolding such that the ligand loop in the mutant no longer constrains Cu(I) as it did in the wild type. These three hydrogen bonds involving the ligand loop assume added significance when one recognizes that a similar hydrogen bonded network does not exist on the opposite side of the metal site where the loop containing the axial methionine ligand is not pinned to the neighboring strand by hydrogen bonds but is only constrained by van der Waals contacts between hydrophobic residues. Strikingly, ligands occupy nearly identical positions even in blue copper proteins whose loops containing these ligands very considerably in length. Proteins with longer ligand loops achieve this structural conservation of ligand geometry by forming tight turns or helical regions through the use of a proline residue and a series of intraloop hydrogen bonds. Similar combinations restrict the conformations of shorter loops. For example, amicyanin forms a cyclic triad of hydrogen bonds between ligand backbones: CysNH–MetCO, MetNH–HisCO and HisNH– CysCO [39]. The analogous triads lengthen upon reduction ˚ [4] and in S-6 A. of Cu(II) in plastocyanin by about 0.1 A ˚ [40]. The bond faecalis pseudoazurin by about 0.3 A between the methionine carbonyl and cysteine amide is conserved in blue copper proteins as a structural element that pins together the beginning and end of the ligand loop. Notably, in the M112H mutant of azurin, protonation of the abnormal histidine axial ligand disrupts this conserved structural feature [41] and the protonated histidine side chain shifts into the space between the two strands. (It is blocked from rotating in the other direction by hydrophobic side chains that normally sandwich the methionine residue.) The protonated histidine side chain also takes the place of the Met amide in forming a hydrogen bond with the Cys carbonyl. Changing proline residues offers a potentially powerful approach to manipulating these conserved structural features which likely play so important a role in modulating the redox properties of the blue copper proteins. We have previously summarized our results along these lines which confirm earlier work. Thus for the P80A and P80I mutants of pseudoazurin from A. faecalis X-ray crystallography

showed that the Cu(I) is free to adopt a trigonal coordination in each of these mutants [40]. Similarly to the N47L plastocyanin and the unfolding intermediate of azurin, the Cu(II) reduction potential of the P80I mutant of pseudoazurin (450 mV vs. NHE) is raised significantly over that of the native protein (270 mV vs. NHE). For the P94A and P94F mutants of P. denitrificans amicyanin (this work) the corresponding reduction potentials are: 380 mV (P94A) and 415 mV (P94F) as compared to 265 mV for the wild type, all vs. NHE (Fig. 2). As there is a lack of independent X-ray structures for the amicyanin mutants, the information on pseudoazurin best serves as comparison. Since they show, within the limits of the precision of these structural determinations, no significant perturbation of the Cu(II) geometry [40] one can infer that there is little change in the energetics of the oxidized state. On the assumption that the solvation contributions accompanying Cu(II) are similar in mutants and wild type, we take our observation that we see an increase of about 45 J / mol K (from 253.368 J / mol K to 28.665 J / mol K) in the P46F mutant to indicate a significantly less constrained structure for the Cu(I) relative to the Cu(II) form of the mutant as compared to the relative Cu(II) and Cu(I) constraints of the wild type. Our observation of a drop in the pKa of the histidine from around 7 in the wild type to less than 5 in the P94F mutant of amicyanin may be due to the formation of a new hydrogen bond. Such hydrogen bond formations have been seen in azurins, and other blue copper proteins that protonate with much lower pKa values when they form a second hydrogen bond to the thiolate the proton of which originates from the amide of the second residue following the cysteine in the amino acid sequence [42]. The crystal structures of oxidized pseudoazurins show a decrease in ˚ (wild type) to the amide–thiolate distance from 4.05 A

Fig. 2. Reduction potentials of P. denitrificans amicyanins as a function of pH (j wild type, s P94A mutant, n P94F mutant).

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˚ (P80I mutant) [40]. A similar decrease in distance 3.64 A characterizes the reduced proteins. Importantly, the participating amide is favorably oriented for hydrogen bond formation. The red shift of the blue absorption band and the larger parallel hyperfine anisotropy in the mutant also suggest the formation of a second hydrogen bond to the thiolate ligand. Another mechanism that has been proposed for controlling the pKa is blocking of solvent access or inhibition of rotation of the imidazole ring by the protein matrix [18,43]. However, the pKa near 6.3 for the P94A mutant of amicyanin argues against steric limitations since the analogous crystal structures show that the active site becomes highly accessible to solvent [40]. One should consider a further possibility. The reduced, unprotonated state is stabilized by 0.15 eV relative to the oxidized state. A similar increase in stability relative to the reduced protonated state would correspond to a pKa decrease of over 2 pH units. X-ray structures show that reduced protonated plastocyanin undergoes a substantial change in coordination with the copper to methionine ˚ as the sulfur distance decreasing from 2.82 to 2.51 A copper moves toward the methionine to become trigonally coordinated to the cysteine thiolate, the histidine and the methionine [16]. This dramatic movement suggests that the structure and stability of the tricoordinate copper, i.e., the protonated state, is not greatly affected by the folded polypeptide. Recently, the ligand loop of Paracoccus versutus amicyanin was replaced with the ligand loop of Thiobacillus ferrooxidans rusticyanin, resulting in a lowering of the pKa from 6.8 (wild type) to less than 4.5 (mutant) [44]. The further observation that the loop switch similarly increased the reduction potential suggests that there may be a tradeoff between optimizing electron transfer function and enhancing imidazole–copper bonding.

4. Conclusion The evidence above and the following discussion strongly support the proposition that the folded protein constrains copper(I) more than it does copper(II) in low-potential blue copper sites. As the constraint of the Cu(I) redox state is relaxed, the potentials increase markedly. Hydrogen bonding networks in the ligand loop play a key role in tuning the reduction potentials. Therefore, removing those bonds that structurally organize the metal binding, loop elevates the Cu(II) / Cu(I) potential. We conclude that potentials around 400 mV are characteristic of a common species: a copper in an unconstrained (His) 2 (Cys)(Met) ligand framework embedded in hydrophobic residues that are less structured than they are in the related wild type proteins with lower redox potentials.

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Acknowledgements This work was supported by the NIH (DK19038 to H.B.G.; GM16424 to J.H.R.; NIH Training Fellowship in Cellular and Molecular Biology to M.C.M.). We thank Hans Freeman and Victor Davidson for very helpful comments on this work.

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