(13)C[(13)C] 2D NMR: a novel strategy for the study of paramagnetic proteins with slow electronic relaxation rates

Oxidized human [2Fe-2S] ferredoxin has a notably slow electronic relaxation rate, which precludes the observation of signals from nuclei near the iron-sulfur cluster by conventional 2D or 3D methods that utilize proton detection. We have demonstrated the utility of (13)C[(13)C]CT-COSY in identifying connectivity information from fast relaxing carbon nuclei near the paramagnetic center, including those from residues that ligate the metal center.     

Strategy for the study of paramagnetic proteins with slow electronic relaxation rates by nmr spectroscopy: application to oxidized human [2Fe-2S] ferredoxin

NMR studies of paramagnetic proteins are hampered by the rapid relaxation of nuclei near the paramagnetic center, which prevents the application of conventional methods to investigations of the most interesting regions of such molecules. This problem is particularly acute in systems with slow electronic relaxation rates. We present a strategy that can be used with a protein with slow electronic relaxation to identify and assign resonances from nuclei near the paramagnetic center. Oxidized human [2Fe-2S] ferredoxin (adrenodoxin) was used to test the approach. The strategy involves six steps: (1) NMR signals from (1)H, (13)C, and (15)N nuclei unaffected or minimally affected by paramagnetic effects are assigned by standard multinuclear two- and three-dimensional (2D and 3D) spectroscopic methods with protein samples labeled uniformly with (13)C and (15)N. (2) The very broad, hyperfine-shifted signals from carbons in the residues that ligate the metal center are classified by amino acid and atom type by selective (13)C labeling and one-dimensional (1D) (13)C NMR spectroscopy. (3) Spin systems involving carbons near the paramagnetic center that are broadened but not hyperfine-shifted are elucidated by (13)C[(13)C] constant time correlation spectroscopy (CT-COSY). (4) Signals from amide nitrogens affected by the paramagnetic center are assigned to amino acid type by selective (15)N labeling and 1D (15)N NMR spectroscopy. (5) Sequence-specific assignments of these carbon and nitrogen signals are determined by 1D (13)C[(15)N] difference decoupling experiments. (6) Signals from (1)H nuclei in these spin systems are assigned by paramagnetic-optimized 2D and 3D (1)H[(13)C] experiments. For oxidized human ferredoxin, this strategy led to assignments (to amino acid and atom type) for 88% of the carbons in the [2Fe-2S] cluster-binding loops (residues 43-58 and 89-94). These included complete carbon spin-system assignments for eight of the 22 residues and partial assignments for each of the others. Sequence-specific assignments were determined for the backbone (15)N signals from nine of the 22 residues and ambiguous assignments for five of the others.     

Inner-sphere reorganization energy of iron-sulfur clusters studied with theoretical methods

Models of several types of iron-sulfur clusters (e.g., Fe(4)S(4)(SCH(3))(4)(2-/3-/4-)) have been studied with the density functional B3LYP method and medium-sized basis sets. In a vacuum, the inner-sphere reorganization energies are 40, 76, 40, 62, 43, and 42 kJ/mol for the rubredoxin, [2Fe-2S] ferredoxin, Rieske, [4Fe-4S] ferredoxin, high-potential iron protein, and desulfoferrodoxin models, respectively. The first two types of clusters were also studied in the protein, where the reorganization energy was approximately halved. This change is caused by the numerous NH.S(Cys) hydrogen bonds to the negatively charged iron-sulfur cluster, giving rise to a polar local environment. The reorganization energy of the iron-sulfur clusters is low because the iron ions retain the same geometry and coordination number in both oxidation states. Cysteine ligands give approximately the same reorganization energy as imidazole, but they have the advantage of stabilizing a lower coordination number and giving more covalent bonds and therefore more effective electron-transfer paths.     

Geometric and electrostatic study of the [4Fe-4S] cluster of adenosine-5'-phosphosulfate reductase from broken symmetry density functional calculations and extended X-ray absorption fine structure spectroscopy

Adenosine-5'-phosphosulfate reductase (APSR) is an iron-sulfur protein that catalyzes the reduction of adenosine-5'-phosphosulfate (APS) to sulfite. APSR coordinates to a [4Fe-4S] cluster via a conserved CC-X(~80)-CXXC motif, and the cluster is essential for catalysis. Despite extensive functional, structural, and spectroscopic studies, the exact role of the iron-sulfur cluster in APS reduction remains unknown. To gain an understanding into the role of the cluster, density functional theory (DFT) analysis and extended X-ray fine structure spectroscopy (EXAFS) have been performed to reveal insights into the coordination, geometry, and electrostatics of the [4Fe-4S] cluster. X-ray absorption near-edge structure (XANES) data confirms that the cluster is in the [4Fe-4S](2+) state in both native and substrate-bound APSR while EXAFS data recorded at ~0.1 ? resolution indicates that there is no significant change in the structure of the [4Fe-4S] cluster between the native and substrate-bound forms of the protein. On the other hand, DFT calculations provide an insight into the subtle differences between the geometry of the cluster in the native and APS-bound forms of APSR. A comparison between models with and without the tandem cysteine pair coordination of the cluster suggests a role for the unique coordination in facilitating a compact geometric structure and "fine-tuning" the electronic structure to prevent reduction of the cluster. Further, calculations using models in which residue Lys144 is mutated to Ala confirm the finding that Lys144 serves as a crucial link in the interactions involving the [4Fe-4S] cluster and APS.     

Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis

The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe-4S] and one [4Fe-4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S](+1/0) (E(0')=-158±5mV); [4Fe-4S](+2/+1) (E(0')=-474±5mV) and [3Fe-4S](0/-2) (E(0')=-660±5mV). The effect of pH on these signals showed that the reduced [3Fe-4S](0) cluster has a pK'(red)(')=5.1±0.1, the [4Fe-4S](+2/+1) centre is pH independent, and the [3Fe-4S](0/-2) reduction is accompanied by the binding of two protons. The ability of the [3Fe-4S](0) cluster to be converted into a new [4Fe-4S] cluster was proven. The redox potential of the original [4Fe-4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70mV) of the redox potential of the original centre. Being most [Fe-S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed.     

Chemical activity of iron in [2Fe-2S]-protein centers and FeS2(100) surfaces

Iron atoms bonded to sulfur play an important role in proteins, heterogeneous catalysts, and gas sensors. First-principles density functional calculations were used to investigate the structure and chemical activity of a unique [2Fe-2S] center in the split-Soret cytochrome c (Ssc) from Desulfovibrio desulfuricans. In agreement with a previously proposed structural model [Abreu et al., J. Biol. Inorg. Chem. 2003, 8, 360], it is found that the [2Fe-2S] cluster is located in a surface pocket of the Ssc and bonded to only three cysteines. The [2Fe-2S] center in the Ssc is nonplanar and somewhat distorted with respect to canonical [2Fe-2S] centers seen in proteins where the iron-sulfur unit is bonded to four cysteines. In the Ssc, the lack of one Fe-cysteine bond is partially compensated by the separation between the cysteines that minimizes electrostatic repulsion among these ligands. The unique structure of the [2Fe-2S] center in the Ssc makes the center more chemically active than canonical [2Fe-2S] centers in proteins, (RS)(4)[2Fe-2S] inorganic complexes, and an FeS2(100) surface. A [2Fe-2S] center in the Ssc interacts efficiently with electron acceptors (O2, NO, CO) and poorly with a Lewis base such as H2O. The interaction with molecular oxygen is so strong that eventually oxidatively destroys the [2Fe-2S] unit. The bonding energy of the ligands to the [2Fe-2S] centers and FeS2(100) surface increases following the sequence: H2O < CO < NO < O2. The higher the electron affinity of the ligand, the larger its bonding energy. A relatively large positive charge on the Fe cations in FeS2(100) makes this sulfide surface less reactive toward O2, CO, and NO than the [2Fe-2S] centers in proteins and inorganic complexes.     

An approach based on quantum chemistry calculations and structural analysis of a [2Fe-2S] ferredoxin that reveal a redox-linked switch in the electron-transfer process to the Fd-NADP+ reductase

[2Fe-2S] ferredoxins act as electron carriers in photosynthesis by mediating the transfer of electrons from photosystem I to various enzymes such as ferredoxin:NADP(+):reductase (FNR). We have analyzed by density functional theory the possible variations of the electronic properties of the [2Fe-2S] ferredoxin, from the cyanobacterium Anabaena, depending on the redox-linked structural changes observed by X-ray diffraction at atomic resolution (Morales, R.; et al. Biochemistry 1999, 38, 15764-15773). The present results point out a specific and concerted role of Ser47, Phe65, and Glu94 located at the molecule surface, close to the iron-sulfur cluster. These residues were already known to be crucial for efficient electron transfer to FNR (e.g., Hurley, J. K.; et al. Biochemistry 1997, 36, 11100-11117). Our calculations suggest that the Glu94 carboxylate negative charge regulates the electron charge delocalization between the Ser47 CO group and the Phe65 aromatic ring, depending on the redox state. The Glu94 carboxylate is stabilized by a strong hydrogen bond implicating a hydroxyl-containing side chain (i.e., Ser or Thr) at location 47. We propose that the Phe65 ring acts as an intermediary carrier receiving the reducing electron prior to its transfer from the reduced Fd to FNR, in view of its central role in the Fd-FNR interaction.     

Theoretical Study on Redox Potential Control of Iron-Sulfur Cluster by Hydrogen Bonds: A Possibility of Redox Potential Programming

The effect of hydrogen bonds around the active site of Anabaena [2Fe-2S] ferredoxin (Fd) on a vertical ionization potential of the reduced state (IP(red)) is examined based on the density functional theory (DFT) calculations. The results indicate that a single hydrogen bond increases the relative stability of the reduced state, and shifts IP(red) to a reductive side by 0.31–0.33 eV, regardless of the attached sulfur atoms. In addition, the IP(red) value can be changed by the number of hydrogen bonds around the active site. The results also suggest that the redox potential of [2Fe-2S] Fd is controlled by the number of hydrogen bonds because IP(red) is considered to be a major factor in the redox potential. Furthermore, there is a possibility that the redox potentials of artificial iron-sulfur clusters can be finely controlled by the number of the hydrogen bonds attached to the sulfur atoms of the cluster.     

Are free radicals involved in IspH catalysis? An EPR and crystallographic investigation

The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and (2)H, (17)O, and (57)Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe-4S](2+) cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate's 3-CH(2)OH group has rotated away from the reduced iron-sulfur cluster, and the next, η(3)-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (4 and 5) follow the same reaction mechanism.     

Electron-nuclear double resonance spectroscopic evidence that S-adenosylmethionine binds in contact with the catalytically active [4Fe-4S](+) cluster of pyruvate formate-lyase activating enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S](+) interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S](+) of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). (2)H and (13)C ENDOR spectroscopy was performed on [4Fe-4S](+)-PFL-AE (S = (1)/(2)) in the presence of AdoMet labeled at the methyl position with either (2)H or (13)C (denoted [1+/AdoMet]). The observation of a substantial (2)H coupling of approximately 1 MHz ( approximately 6-7 MHz for (1)H), as well as hyperfine-split signals from the (13)C, manifestly require that AdoMet lie close to the cluster. (2)H and (13)C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S](2+) state of PFL-AE, which is visualized through cryoreduction of the frozen [4Fe-4S](2+)/AdoMet complex to form the reduced state (denoted [2+/AdoMet](red)) trapped in the structure of the oxidized state. (2)H and (13)C ENDOR spectra for [2+/AdoMet](red) are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Analysis of 2D field-frequency (13)C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the (2)H and (13)C ENDOR, we have estimated the distance from the closest methyl proton of AdoMet to the closest iron of the cluster to be approximately 3.0-3.8 A, while the distance from the methyl carbon to the nearest iron is approximately 4-5 A. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S](2+/+) cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.     

Mechanistic insight into the nitrosylation of the [4Fe-4S] cluster of WhiB-like proteins

The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO → 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.     

Ligand K-edge X-ray absorption spectroscopy of [Fe4S4]1+,2+,3+ clusters: changes in bonding and electronic relaxation upon redox

Sulfur K-edge X-ray absorption spectroscopy (XAS) is reported for [Fe(4)S(4)](1+,2+,3+) clusters. The results are quantitatively and qualitatively compared with DFT calculations. The change in covalency upon redox in both the [Fe(4)S(4)](1+/2+) (ferredoxin) and the [Fe(4)S(4)](2+/3+) (HiPIP) couple are much larger than that expected from just the change in number of 3d holes. Moreover, the change in the HiPIP couple is higher than that of the ferredoxin couple. These changes in electronic structure are analyzed using DFT calculations in terms of contributions from the nature of the redox active molecular orbital (RAMO) and electronic relaxation. The results indicate that the RAMO of HiPIP has 50% ligand character, and hence, the HiPIP redox couple involves limited electronic relaxation. Alternatively, the RAMO of the ferredoxin couple is metal-based, and the ferredoxin redox couple involves extensive electronic relaxation. The contributions of these RAMO differences to ET processes in the different proteins are discussed.     

Characterization of iron dinitrosyl species formed in the reaction of nitric oxide with a biological Rieske center

Reactions of nitric oxide with cysteine-ligated iron-sulfur cluster proteins typically result in disassembly of the iron-sulfur core and formation of dinitrosyl iron complexes (DNICs). Here we report the first evidence that DNICs also form in the reaction of NO with Rieske-type [2Fe-2S] clusters. Upon treatment of a Rieske protein, component C of toluene/o-xylene monooxygenase from Pseudomonas sp. OX1, with an excess of NO(g) or NO-generators S-nitroso-N-acetyl-D,L-pencillamine and diethylamine NONOate, the absorbance bands of the [2Fe-2S] cluster are extinguished and replaced by a new feature that slowly grows in at 367 nm. Analysis of the reaction products by electron paramagnetic resonance, M?ssbauer, and nuclear resonance vibrational spectroscopy reveals that the primary product of the reaction is a thiolate-bridged diiron tetranitrosyl species, [Fe(2)(μ-SCys)(2)(NO)(4)], having a Roussin's red ester (RRE) formula, and that mononuclear DNICs account for only a minor fraction of nitrosylated iron. Reduction of this RRE reaction product with sodium dithionite produces the one-electron-reduced RRE, having absorptions at 640 and 960 nm. These results demonstrate that NO reacts readily with a Rieske center in a protein and suggest that dinuclear RRE species, not mononuclear DNICs, may be the primary iron dinitrosyl species responsible for the pathological and physiological effects of nitric oxide in such systems in biology.     

Spectroscopic approaches to elucidating novel iron-sulfur chemistry in the "radical-Sam" protein superfamily

Electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and M?ssbauer spectroscopies and other physical methods have provided important new insights into the radical-SAM superfamily of proteins, which use iron-sulfur clusters and S-adenosylmethionine to initiate H atom abstraction reactions. This remarkable chemistry involves the generation of the extremely reactive 5'-deoxyadenosyl radical, the same radical intermediate utilized in B12-dependent reactions. Although early speculation focused on the possibility of an organometallic intermediate in radical-SAM reactions, current evidence points to novel chemistry involving a site-differentiated [4Fe-4S] cluster. The focus of this forum article is on one member of the radical-SAM superfamily, pyruvate formate-lyase activating enzyme, and how physical methods, primarily EPR and ENDOR spectroscopies, are contributing to our understanding of its structure and mechanism. New ENDOR data supporting coordination of the methionine moiety of SAM to the unique site of the [4Fe-4S]2+/+ cluster are presented.     

Mechanism on two-electron oxidation of ubiquinol at the Qp site in cytochrome bc1 complex: B3LYP study with broken symmetry

The molecular and electronic structures of the Rieske iron-sulfur [2Fe-2S] cluster with an imidazolate and imidazole were investigated by using usual unrestricted and broken symmetry B3LYP methods for the highest and lowest spin states, respectively. The electronic structures of the lowest spin states were determined by the spin contaminations and natural orbital analyses. It was shown that the spin contamination presents the number of pairs of the antiferromagnetic spin couplings. The oxidation mechanism of the ubquinol at the Q(p) site of the cytochrome bc(1) complex was also examined by the broken symmetry B3LYP methods. In the [2Fe-2S] clusters with an imidazolate, the oxidized and lowest spin state, [(Imz(-))FeS](ox)LS, was lowest in energy among four possible states, consistent with experimental observations. In the examination of the mechanism of the ubquinol oxidation, it was confirmed that the ubiquinol docks between the imidazolate of [2Fe-2S] clusters and Glu272(-) of cytochrome b by the hydrogen bonds before the oxidation proceeds, consistent with the experimental proposals. Our results support a "Glu272-first" mechanism that Glu272 serves as an acceptor of the first proton from the ubiquinol and subsequently the proton-coupled electron transfer (PCET) occurs from the ubisemiquinone anion to the Rieske iron-sulfur [2Fe-2S] cluster.     

Light-induced charge separation across the photosynthetic membrane: a proposed structure for the photosystem I reaction center

The photosystem I reaction center is reviewed from the standpoint of electron acceptors, polypeptide composition, and structural organization. Experimental difficulties in the current model are highlighted, and current results on the polypeptide location of each acceptor are reviewed. The amount of iron and labile sulfide, the types of iron-sulfur clusters, and the number of ∼ 64-kDa polypeptides are considered in light of a model for the reaction center. In particular, the presence of [2Fe-2S] clusters in photosystem I has significance in the structure and organization of F_x on the reaction center. Since four cysteinyl ligands are assumed to hold an iron-sulfur cluster, a photosystem I subunit may consist of two ∼ 64-kDa polypeptides bridged by a [2Fe-2S] cluster. The reaction center would consist of a symmetrical pair of these subunits positioned so that two [2Fe-2S] clusters are in magnetic interaction, thereby constituting F_x. The reaction center core would therefore incorporate four ∼ 64-kDa polypeptides and have a molecular weight in excess of 250 kDa     

Correlation between the magnetic g tensors and the local cysteine geometries for a series of reduced [2Fe-2S*] protein clusters. A quantum chemical density functional theory and structural analysis

We relied on the density functional theory (DFT) to study the electronic structure of the [2Fe-2S*](SH)4 model of the active site of 2Fe ferredoxins and other proteins containing reduced [2Fe-2S*] clusters. The two (Fe(3+)-Fe(2+)-S-H) dihedral angles Omega1 and Omega2 defined for the two ligands on the ferrous side were allowed to vary, while the two other (Fe(2+)-Fe(3+)-S-H) angles Omega3 and Omega4 on the ferric side were kept constant. The Landé (g), magnetic hyperfine, and quadrupole tensors for two geometries, C2 (Omega1 = Omega2) and Cs (Omega1 = -Omega2), were calculated. To apply our model to the actual proteins, we listed all of the crystallographic structures available for the [2Fe-2S*] systems. A classification of these proteins, based on the four dihedral angles [Omega(i)](i=1-4), separates them into three main classes. The main structural feature of the first class (Omega1 approximately Omega2), with an average dihedral angle Omega(av) = (Omega1 + Omega2)/2 comprised between 115 degrees and 150 degrees, corresponds to a local ferrous C2 geometry (rather than C2nu, as previously assumed by Bertrand and Gayda: Biochim. Biophys. Acta 1979, 579, 107). We then established a direct correlation between the three principal g values and Omega(av). It is the first time that such a link has been made between the spectroscopic and structural parameters, a link, moreover, fully rationalized by our DFT calculations. We finally point out the basic differences between our C2 results with those of the C2nu phenomenological model proposed in the late 1970s by Bertrand and Gayda.     

Rieske protein from Thermus thermophilus: 15N NMR titration study demonstrates the role of iron-ligated histidines in the pH dependence of the reduction potential

A unique feature of Rieske proteins is the pH dependence of their reduction potentials. It has been proposed that protonation of the Nepsilon2 atoms of the two histidine rings ligated to the iron-sulfur cluster is coupled with cluster reduction (electron transfer). We have incorporated [15Ndelta1, 15Nepsilon2]-histidine into the Rieske protein from Thermus thermophilis and have used 15N NMR spectroscopy to determine the pKa values of the histidine residues in the oxidized state of the protein. As expected from studies of a Rieske-type ferredoxin, the signals from the 15Ndelta1 atoms directly bound to iron were too broad to be detected, but broad signals could be detected from the 15Nepsilon2 atom of each of the ligated histidine rings. We measured the chemical shifts of these signals as a function of pH between pH 6 and pH 12 and fitted them to theoretical titration curves. The results yielded well-separated pKa values for the two histidines (7.46 and 9.24), with Hill coefficients close to unity. The pKa values are in excellent agreement with values predicted from the pH dependence of the reduction potentials (7.85 and 9.65).