柳澤 幸子

Synechocystis PCC 6803 has a high demand for iron (10 times greater than Escherichia coli) to sustain photosynthesis and is unusual in possessing at least two putative iron-binding proteins of a type normally associated with ATP-binding cassette-type importers. It has been suggested that one of these, FutA2, binds ferrous iron, but herein we clearly demonstrate that this protein avidly binds Fe(III), the oxidation state preference of periplasmic iron-binding proteins. Structures of apo-FutA2 and Fe-FutA2 have been determined at 1.7 and 2.7 A, respectively. The metal ion is bound in a distorted trigonal bipyramidal arrangement with no exogenous anions as ligands. The metal-binding environment, including the second coordination sphere and charge properties, is consistent with a preference for Fe( III). Atypically, FutA2 has a Tat signal peptide, and its inability to coordinate divalent cations may be crucial to prevent metals from binding to the folded protein prior to export from the cytosol. A loop containing the His43 ligand undergoes considerable movement in apo-versus Fe-FutA2 and may control metal release to the importer. Although these data are consistent with FutA2 being the periplasmic component involved in iron uptake, deletion of another putative ferric binding protein, FutA1, has a greater effect on the accumulation of iron and is more analogous to a Delta futA1 Delta futA2 double mutant than Delta futA2. Here, we also discover that there is a reduced level of ferric FutA2 in the periplasm of the Delta futA1 mutant providing an explanation for its severe iron-uptake phenotype.

Atomic resolution structures of the pseudoazurin (PAZ) variant into which the shorter ligand-containing loop of amicyanin (AMI) is introduced have been determined. The mutated loop adopts a different conformation in PAZAMI than in AMI. The copper site structure is affected, with the major influence being an increased axial interaction resulting in the shortest Cu(II)-S(Met) bond observed for the cupredoxin family of electron-transfer proteins. This is accompanied by a lengthening of the important Cu-S(Cys) bond and enhanced tetragonal distortion, consistent with the influence of the PAZAMI loop contraction on the UV/vis spectrum. The change in active site geometry is the major cause of the 50 mV decrease in reduction potential. The copper site structure changes very little upon reduction, consistent with the distorted site still possessing the properties required to facilitate rapid electron transfer. The exposed His ligand on the loop protonates in the reduced protein and reasons for the increased pK(a) compared to that of PAZ are discussed. The area surrounding the His ligand is more hydrophobic in PAZAMI than in PAZ, while electron self-exchange, which involves homodimer formation via this surface patch, is decreased. The nature of the side chains in this region, as dictated by the sequence of the ligand-containing loop, is a more significant factor than hydrophobicity for facilitating transient protein interactions in PAZ. The structure of PAZAMI demonstrates the importance of loop-scaffold interactions for metal sites in proteins.

The thermodynamics of the alkaline transition which influences the spectral and redox properties of the type 1 copper center in phytocyanins has been determined spectroscopically. The proteins investigated include Rhus vernicifera stellacyanin, cucumber basic protein and its Met89Gln variant, and umecyanin, the stellacyanin from horseradish roots, along with its Gln95Met variant. The changes in reaction enthalpy and entropy within the protein series show partial compensatory behavior. Thus, the reaction free energy change (hence the pK (a) value) is rather variable. This indicates that species-dependent differences in reaction thermodynamics, although containing an important contribution from changes in the hydrogen-bonding network of water molecules in the hydration sphere of the protein (which feature enthalpy-entropy compensation), are to a large extent protein-based. The data for axial ligand variants are consistent with the hypothesis of a copper-binding His as the deprotonating residue responsible for this transition.

Periplasmic substrate binding proteins are known for iron, zinc, manganese, nickel, and molybdenum but not copper. Synechocystis PCC 6803 requires copper for thylakoid-localized plastocyanin and cytochrome oxidase. Here we show that mutants deficient in a periplasmic substrate binding protein FutA2 have low cytochrome oxidase activity and produce cytochrome c(6) when grown under copper conditions (150 nM) in which wild-type cells use plastocyanin rather than cytochrome c6. Anaerobic separation of extracts by two-dimensional native liquid chromatography followed by metal analysis and peptide mass-fingerprinting establish that accumulation of copper-plastocyanin is impaired, but iron-ferredoxin is unaffected in Delta futA2 grown in 150 nM copper. However, recombinant FutA2 binds iron in preference to copper in vitro with an apparent Fe(III) affinity similar to that of its paralog FutA1, the principal substrate binding protein for iron import. FutA2 is also associated with iron and not copper in periplasm extracts, and this Fe(III)-protein complex is absent in Delta futA2. There are differences in the soluble protein and small-molecule complexes of copper and iron, and the total amount of both elements increases in periplasm extracts of Delta futA2 relative to wild type. Changes in periplasm protein and small-molecule complexes for other metals are also observed in Delta futA2. It is proposed that FutA2 contributes to metal partitioning in the periplasm by sequestering Fe(III), which limits aberrant Fe(III) associations with vital binding sites for other metals, including copper.

The Phe114Pro mutation to the cupredoxin azurin (AZ) leads to a number of structural changes at the active site attributed to deletion of one of the hydrogen bonds to the Cys112 ligand, removal of the bulky phenyl group from the hydrophobic patch of the protein, and steric interactions made by the introduced Pro. The remaining hydrogen bond between the coordinating thiolate and the backbone amide of Asn47 is strengthened. At the type-1 copper site, the Cu-II-O(Gly45) axial interaction decreases, while the metal moves out of the plane formed by the equatorial His46, Cys112, and His117 ligands, shortening the bond to the axially coordinating Met121. The resulting distorted tetrahedral geometry is distinct from the trigonal bipyramidal arrangement in the wild-type (WT) protein. The unique position of the main S(Cys) -> CuII ligand-to-metal charge-transfer transition in AZ (628 nm) has shifted in the Phe114Pro variant to a value that is more typical for cupredoxins (599 nm). This probably occurs because of the removal of the Phe114Cys112 hydrogen bond. The Phe114Pro mutation results in a 90 mV decrease in the reduction potential of AZ, and removal of the second hydrogen bond to the Cys ligand seems to be the major cause of this change. The C-terminal His117 ligand does not protonate in the reduced Phe114Pro AZ variant, which suggests that none of the structural features altered by the mutation are responsible for the absence of this effect in the WT protein. Upon reduction, the copper displaces further from the equatorial ligand plane and the Cu-S(Met121) bond length decreases. These changes are larger than those seen in the WT protein and contribute to the order of magnitude decrease in the intrinsic electron-transfer capabilities of the Phe114Pro variant.

The main active-site loop of the copper-binding protein azurin (a cupredoxin) has been shortened from C(112)TFPGH(117)SALM(121) to (CTPHPFM118)-T-112-P-115 (the native loop from the cupredoxin amicyanin) and also to (CTPHPM117)-T-112-P-115. The Cu(II) site structure is almost unaffected by shortening, as is that of the Cu(I) center at alkaline pH in the variant with the (CTPHPM117)-T-112-P-115 loop sequence. Subtle spectroscopic differences due to alterations in the spin density distribution at the Cu(II) site can be attributed mainly to changes in the hydrogen-bonding pattern. Electron transfer is almost unaffected by the introduction of the (CTPHPFM118)-T-112-P-115 loop, but removal of the Phe residue has a sizable effect on reactivity, probably because of diminished homodimer formation. At mildly acidic pH values, the His-115 ligand protonates and dissociates from the cuprous ion, an effect that has a dramatic influence on the reactivity of cupredoxins. These studies demonstrate that the amicyanin loop adopts a conformation identical to that found in the native protein when introduced into azurin, that a shorter than naturally occurring C-terminal active-site loop can support a functional T1 copper site, that CTPHPM is the minimal loop length required for binding this ubiquitous electron transfer center, and that the length and sequence of a metal-binding loop regulates a range of structural and functional features of the active site of a metalloprotein.

Type 1 (T1) copper sites promote biological electron transfer (ET) and typically possess a weakly coordinated thioether sulfur from an axial Met [Cu(II)-S(delta)similar to 2.6 to 3.3 angstrom) along with the conserved HiS(2)Cys equatorial ligands. A strong axial bond (Cu(II)-O(epsilon 1)similar to 2.2 angstrom] is sometimes provided by a Gln (as in the stellacyanins), and the axial ligand can be absent (a Val, Leu or Phe in the axial position) as in ceruloplasmin, Fet3p, fungal laccases and some plantacyanins (PLTs). Cucumber basic protein (CBP) is a PLT which has a relatively short Cu(II)-S(Met89) axial bond (2.6 angstrom). The Met89Gln variant of CBP has an electron self-exchange (ESE) rate constant (k(ese), a measure of intrinsic ET reactivity) similar to 7 times lower than that of the wild-type protein. The Met89Val mutation to CBP results in a 2-fold increase in k(ese). As the axial interaction decreases from strong 061 of Gln to relatively weak S-delta of Met to no ligand (Val), ESE reactivity is therefore enhanced by similar to 1 order of magnitude while the reduction potential increases by similar to 350 mV. The variable coordination position at this ubiquitous ET site provides a mechanism for tuning the driving force to optimize ET with the correct partner without significantly compromising intrinsic reactivity. The enhanced reactivity of a three-coordinate T1 copper site will facilitate intramolecular ET in fungal laccases and Fet3p.

The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyanins, plantacyanins, and uclacyanins. All of these proteins possess the typical type 1 His(2)Cys equatorial ligand set at their mononuclear copper sites, but the stellacyanins have an axial Gln ligand in place of the weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. The stellacyanins exhibit altered visible, EPR, and paramagnetic H-1 NMR spectra at elevated pH values and also modified reduction potentials. This alkaline transition occurs with a pK(a) of similar to10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study we demonstrate that the alkaline transition has a similar influence on the visible, EPR, and paramagnetic NMR spectra of cucumber basic protein (CBP), which is a plantacyanin. The mutation of the axial Gln95 ligand into a Met in umecyanin (UMC), the stellacyanin from horseradish roots, and the axial Met89 into a Gln in CBP have very limited, yet similar, influence on the pK(a) for the alkaline transition as judged from alterations in visible spectra. The complete removal of the axial ligand in the Met89Val variant of CBP results in a slightly larger decrease in the pKa for this effect, but similar spectral alterations are still observed at elevated pH. Thus, the axial Gln ligand is not the cause of the alkaline transition in Cu(II) stellacyanins, and alterations in the active site structures of the phytocyanins have a limited effect on this feature. The conserved Lys residue found adjacent to the axial ligand in the sequences of all phytocyanins, and implicated as the trigger for the alkaline transition, has been mutated to an Arg in UMC. The influence of increasing pH on the spectroscopic properties of Lys96Arg UMC is almost identical to those of the wild type protein, and thus, this residue is not responsible for the alkaline transition. However, a positively charged residue in this position seems to be important for the correct folding of UMC. Other possible triggers for the effects seen in the phytocyanins at elevated pH are discussed along with the relevance of the alkaline transition.

The shortest known type 1 copper binding loop (that of amicyanin, Ami) has been introduced into three different cupredoxin beta-barrel scaffolds. All of the loop-contraction variants possess copper centers with authentic type 1 properties and are redox active. The Cu(II) and Co(II) sites experience only small structural alterations upon loop contraction with the largest changes in the azurin variant (AzAmi), which can be ascribed to the removal of a hydrogen bond to the coordinating thiolate sulfur of the Cys ligand. In all cases, loop contraction leads to an increase in the pK(a) of the His ligand found on the loop in the reduced proteins, and in the pseudoazurin (Paz) and plastocyanin (PC) variants the values are almost identical to that of Ami (similar to6.7). Thus, in Paz, PC, and Ami, the length of this loop tunes the pK(a), of the His ligand. In the AzAmi variant, the pK(a) is 5.5, which is considerably higher than the estimated value for Az (<2), and other controlling factors, along with loop length, are involved. The reduction potentials of the loop-contraction variants are all lower than those of the wild-type proteins by similar to30-60 mV, and thus this property of a type 1 copper site is fine-tuned by the C-terminal loop. The electron self-exchange rate constant of Paz is significantly diminished by the introduction of a shorter loop. However, in PcAmi only a 2-fold decrease is observed and in AzAmi there is no effect, and thus in these two cupredoxins loop contraction does not significantly influence electron-transfer reactivity. Loop contraction provides an active site environment in all of the cupredoxins which is preferable for Cu(II), whereas previous loop elongation experiments always favored the cuprous site. Thus, the ligand-containing loop plays an important role in tuning the entatic nature of a type 1 copper center.