Characterization of an autoreduction pathway for the [Fe4S4]3+ cluster of mutant Chromatium vinosum high-potential iron proteins. Site-directed mutagenesis studies to probe the role of phenylalanine 66 in defining the stability of the [Fe4S4] center provide evidence for oxidative degradation via a [Fe3S4] cluster

A number of point mutations of the conserved aromatic residue phenylalanine 66 (Phe66Tyr, -Asn, -Cys, -Ser) in Chromatium vinosum high-potential iron sulfur protein have been examined with the aim of understanding the functional role of this residue. Nonconservative replacements with polar residues have a minimal effect on the midpoint potential of the [Fe4S4]3+/2+ cluster, typically < +25 mV, with a maximum change of +40 mV for Phe66Asn. With the exception of the Phe66Tyr mutant, the oxidized state was found to be unstable relative to the recombinant native, with regeneration of the reduced state. The pathway for this transformation involves degradation of the cluster in a fraction of the sample, which provides the reducing equivalents required to bring about reduction of the remainder of the sample. This degradative reaction proceeds through a transient [Fe3S4]+ intermediate that is characterized by typical g values and power saturation behavior and is prompted by the increased solvent accessibility of the cluster core in the nonconservative Phe66 mutants as evidenced by 1H-15N HMQC NMR experiments. These results are consistent with a model where the critical role of the aromatic residues in the high-potential iron proteins is to protect the cluster from hydrolytic degradation in the oxidized state.     

Delta T 14/Delta D 15 Azotobacter vinelandii ferredoxin I: creation of a new CysXXCysXXCys motif that ligates a [4Fe-4S] cluster

In clostridial-type ferredoxins, each of the two [4Fe-4S]2+/+ clusters receives three of its four ligands from a CysXXCysXXCys motif. Azotobacter vinelandii ferredoxin I (AvFdI) is a seven-iron ferredoxin that contains one [4Fe-4S]2+/+ cluster and one [3Fe-4S]+/0 cluster. During the evolution of the 7Fe azotobacter-type ferredoxins from the 8Fe clostridial-type ferredoxins, one of the two motifs present changed to a CysXXCysXXXXCys motif, resulting in the inability to form a 4Fe cluster and the appearance of a 3Fe cluster in that position. In a previous study, we were unsuccessful in using structure as a guide in designing a 4Fe cluster in the 3Fe cluster position of AvFdI. In this study, we have reversed part of the evolutionary process by deleting two residues between the second and third cysteines. UV/Vis, CD, and EPR spectroscopies and direct electrochemical studies of the purified protein reveal that this DeltaT14/DeltaD15 FdI variant is an 8Fe protein containing two [4Fe-4S]2+/+ clusters with reduction potentials of -466 and -612 mV versus SHE. Whole-cell EPR shows that the protein is present as an 8Fe protein in vivo. These data strongly suggest that it is the sequence motif rather than the exact sequence or the structure that is critical for the assembly of a 4Fe cluster in that region of the protein. The new oxygen-sensitive 4Fe cluster was converted in partial yield to a 3Fe cluster. In known ferredoxins and enzymes that contain reversibly interconvertible [4Fe-4S]2+/+ and [3Fe-4S]+/0 clusters, the 3Fe form always has a reduction potential ca. 200 mV more positive than the 4Fe cluster in the same position. In contrast, for DeltaT14/DeltaD15 FdI, the 3Fe and 4Fe clusters in the same location have extremely similar reduction potentials.     

Redox properties of Desulfovibrio gigas [Fe3S4] and [Fe4S4] ferredoxins and heterometal cubane-type clusters formed within the [Fe3S4] core. Square wave voltammetric studies

The same polypeptide chain (58 amino acids, 6 cysteines) is used to build up two ferredoxins in Desulfovibrio gigas a sulfate reducing organism. Ferredoxin II (FdII) contains a single [Fe3S4] core and ferredoxin I (FdI) mainly a [Fe4S4] core. The [Fe3S4] core can readily be interconverted into a [Fe4S4] complex (J.J.G. Moura, I. Moura, T.A. Kent, J.D. Lipscomb, B.H. Huynh, J. LeGall, A.V. Xavier, and E. Munck, J. Biol. Chem. 257, 6259 (1982)). This interconversion process suggested that the [Fe3S4] core could be used as a synthetic precursor for the formation of heterometal clusters. Co, Zn, Cd, and Ni derivatives were produced (I. Moura, J.J.G. Moura, E. Munck, V. Papaephthymiou, and J. LeGall, J. Am. Chem. Soc. 108, 349 (1986), K. Sureurs, E. Munck, I. Moura, J.J.G. Moura, and J. LeGall, J. Am. Chem. Soc. 109, 3805 (1986), and A.L. Macedo, I. Moura, J.J.G. Moura, K. Surerus, and E. Munck, unpublished results). The redox properties of a series of heterometal clusters (MFe3S4] are assessed using direct electrochemistry (square wave voltammetry--SWV) promoted by Mg(II) at a glassy carbon electrode (derivatives: Cd (-495 mV), Fe (-420 mV), Ni (-360 mV), and Co (-245 mV) vs normal hydrogen electrode (NHE)). In parallel, the electrochemical behavior (cyclic voltammetry--CV, differential pulse voltammetry--DPV and SWV) of FdI and FdII were investigated as well as the cluster interconversion process. In addition to the +1/0 (3Fe cluster) and +2/+1 (4Fe cluster) redox transitions, a very negative redox step, at -690 mV, was detected for the 3Fe core, reminiscent of a postulated further 2e- reduction step, as proposed for D. africanus ferredoxin III by F.A. Armstrong, S.J. George, R. Cammack, E.C. Hatchikian, and A.J. Thomson, Biochem. J. 264, 265 (1989). The electrochemical redox potential values are compared with those determined by independent methods (namely by electron paramagnetic resonance (EPR) and visible spectroscopy).     

The role and properties of the iron-sulfur cluster in Escherichia coli dihydroxy-acid dehydratase

Dihydroxy-acid dehydratase has been purified from Escherichia coli and characterized as a homodimer with a subunit molecular weight of 66,000. The combination of UV visible absorption, EPR, magnetic circular dichroism, and resonance Raman spectroscopies indicates that the native enzyme contains a [4Fe-4S]2+,+ cluster, in contrast to spinach dihydroxy-acid dehydratase which contains a [2Fe-2S]2+,+ cluster (Flint, D. H., and Emptage, M. H. (1988) J. Biol. Chem. 263, 3558-3564). In frozen solution, the reduced [4Fe-4S]+ cluster has a S = 3/2 ground state with minor contributions from forms with S = 1/2 and possibly S = 5/2 ground states. Resonance Raman studies of the [4Fe-4S]2+ cluster in E. coli dihydroxy-acid dehydratase indicate non-cysteinyl coordination of a specific iron, which suggests that it is likely to be directly involved in catalysis as is the case with aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4674-4678). Dihydroxy-acid dehydratase from E. coli is inactivated by O2 in vitro and in vivo as a result of oxidative degradation of the [4Fe-4S]cluster. Compared to aconitase, the oxidized cluster of E. coli dihydroxy-acid dehydratase appears to be less stable as either a cubic or linear [3Fe-4S] cluster or a [2Fe-2S] cluster. Oxidative degradation appears to lead to a complete breakdown of the Fe-S cluster, and the resulting protein cannot be reactivated with Fe2+ and thiol reducing agents.     

Spectroelectrochemical characterization of the metal centers in carbon monoxide dehydrogenase (CODH) and nickel-deficient CODH from Rhodospirillum rubrum

Carbon-monoxide dehydrogenase (CODH) from Rhodospirillum rubrum contains two metal centers: a Ni-X-[Fe4S4]2+/1+ cluster (C-center) that serves as the COoxidation site and a standard [Fe4S4]2+/1+ cluster (B-center) that mediates electron flow from the C-center to external electron acceptors. Four states of the C-center were previously identified in electron paramagnetic resonance (EPR) and M?ssbauer studies. In this report, EPR-redox titrations demonstrate that the fully oxidized, diamagnetic form of the C-center (Cox) undergoes a one-electron reduction to the Cred1 state (gav = 1.87) with a midpoint potential of -110 mV. The reduction of Cox to Cred1 is shown to coincide with the reduction of an [Fe4S4]2+/1+ cluster in redox-titration experiments monitored by UV-visible spectroscopy. Nickel-deficient CODH, which is devoid of nickel yet contains both [Fe4S4]2+/1+ clusters, does not exhibit EPR-active states or reduced Fe4S4 clusters at potentials more positive than -350 mV.     

3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.     

A hydrophobic cluster between transmembrane helices 5 and 6 constrains the thyrotropin-releasing hormone receptor in an inactive conformation

We have studied the role of a highly conserved tryptophan and other aromatic residues of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) that are predicted by computer modeling to form a hydrophobic cluster between transmembrane helix (TM)5 and TM6. The affinity of a mutant TRH-R, in which Trp279 was substituted by alanine (W279A TRH-R), for most tested agonists was higher than that of wild-type (WT) TRH-R, whereas its affinity for inverse agonists was diminished, suggesting that W279A TRH-R is constitutively active. We found that W279A TRH-R exhibited 3.9-fold more signaling activity than WT TRH-R in the absence of agonist. This increased basal activity was inhibited by the inverse agonist midazolam, confirming that the mutant receptor is constitutively active. Computer-simulated models of the unoccupied WT TRH-R, the TRH-occupied WT TRH-R, and various TRH-R mutants predict that a hydrophobic cluster of residues, including Trp279 (TM6), Tyr282, and Phe199 (TM5), constrains the receptor in an inactive conformation. In support of this model, we found that substitution of Phe199 by alanine or of Tyr282 by alanine or phenylalanine, but not of Tyr200 (by alanine or phenylalanine), resulted in a constitutively active receptor. We propose that a hydrophobic cluster including residues in TM5 and TM6 constrains the TRH-R in an inactive conformation via interhelical interactions. Disruption of these constraints by TRH binding or by mutation leads to changes in the relative positions of TM5 and TM6 and to the formation of an active form of TRH-R.     

Modified ligands to FA and FB in photosystem I. Proposed chemical rescue of a [4Fe-4S] cluster with an external thiolate in alanine, glycine, and serine mutants of PsaC

The FB and FA electron acceptors in Photosystem I (PS I) are [4Fe-4S] clusters ligated by cysteines provided by PsaC. In a previous study (Mehari, T., Qiao, F., Scott, M. P., Nellis, D., Zhao, J., Bryant, D., and Golbeck, J. H. (1995) J. Biol. Chem. 270, 28108-28117), we showed that when cysteines 14 and 51 were replaced with serine or alanine, the free proteins contained a S = 1/2, [4Fe-4S] cluster at the unmodified site and a mixed population of S = 1/2, [3Fe-4S] and S = 3/2, [4Fe-4S] clusters at the modified site. We show here that these mutant PsaC proteins can be rebound to P700-FX cores, resulting in fully functional PS I complexes. The low temperature EPR spectra of the C14XPsaC.PS I complexes (where X = S, A, or G) show the photoreduction of a wild-type FA cluster and a modified FB' cluster, the latter with g values of 2.115, 1.899, and 1.852 and linewidths of 110, 70, and 85 MHz. Since neither alanine nor glycine contains a suitable side group, an external thiolate provided by beta-mercaptoethanol has likely been recruited to supply the requisite ligand to the [4Fe-4S] cluster. The EPR spectrum of the C51SPsaC.PS I complex differs from that of the C51APsaC.PS I or C51GPsaC.PS I complexes by the presence of an additional set of resonances, which may be derived from the serine oxygen-ligated cluster. In all other mutant PS I complexes, a wild-type spin-coupled interaction spectrum appears when FA and FB are simultaneously reduced. Single turnover flash studies indicate approximately 50% efficient electron transfer to FA/FB in the C14SPsaC.PS I, C51SPsaC.PS I, C14GPsaC.PS I, and C51GPsaC.PS I mutants and less than 40% in the C14APsaC.PS I and C51APsaC.PS I mutants, compared with approximately 76% in the PS I core reconstructed with wild-type PsaC. These data are consistent with the measurements of the rates of cytochrome c6-NADP+ reductase activity, indicating lower rates in the alanine mutants. It is proposed that the chemical rescue of a [4Fe-4S] cluster with a recruited external thiolate at the modified site allows the mutant PsaC proteins to rebind to PS I and to function in forward electron transfer.     

Phenylketonuria. The in vivo hydroxylation rate of phenylalanine into tyrosine is decreased

In phenylketonuria (PKU), the enzyme phenylalanine hydroxylase is deficient, resulting in a decreased conversion of phenylalanine (Phe) into tyrosine (Tyr). The severity of the disease is expressed as the tolerance for Phe at 5 yr of age. In PKU patients it is assumed that the decreased conversion of Phe into Tyr is directly correlated with the tolerance for Phe. We investigated this correlation by an in vivo stable isotope study. The in vivo residual hydroxylation was quantitated using a primed continuous infusion of L-[ring- 2H5]Phe and L-[1-13C]Tyr and the determination of the isotopic enrichments of L-[ring-2H5]Phe, L-[ring-2H4]Tyr, and L-[1-13C]Tyr in plasma. Previous reports by Thompson and coworkers (Thompson, G.N., and D. Halliday. 1990. J. Clin. Invest. 86:317-322; Thompson, G.N., J.H. Walter, J.V. Leonard, and D. Halliday. 1990. Metabolism. 39:799-807; Treacy, E., J.J. Pitt, K. Seller, G.N. Thompson, S. Ramus, and R.G.H. Cotton. 1996. J. Inherited Metab. Dis. 19:595- 602), applying the same technique, showed normal in vivo hydroxylation rates of Phe in almost all PKU patients. Therefore, our study was divided up in two parts. First, the method was re-evaluated. Second, the correlation between the in vivo hydroxylation of Phe and the tolerance for Phe was tested in seven classical PKU patients. Very low (0.13- 0.95 micromol/kg per hour) and normal (4.11 and 6.33 micromol/kg per hour) conversion rates were found in patients and controls, respectively. Performing the infusion study twice in the same patient and wash-out studies of the labels at the end of the experiment in a patient and control showed that the method is applicable in PKU patients and gives consistent data. No significant correlation was observed between the in vivo hydroxylation rates and the tolerances. The results of this study, therefore, showed that within the group of patients with classical PKU, the tolerance does not depend on the in vivo hydroxylation.     

Residues Val254, His256, and Phe259 of the angiotensin II AT1 receptor are not involved in ligand binding but participate in signal transduction

The role of the external third of helix VI of the angiotensin II (AII) AT1 receptor for the interaction with its ligand and for the subsequent signal transduction was investigated by individually replacing residues 252-256 by Ala, and residues 259 or 261 by Tyr, and permanently transfecting the resulting mutants to Chinese hamster ovary (CHO) cells. Binding experiments showed no great changes in affinity of any of the mutants for AII, [Sar1]-AII, or [Sar1, Leu8]-AII, but the affinity for the nonpeptide antagonist DuP753 was significantly decreased. The inositol phosphate response to AII was remarkably decreased in mutants V254A, H256A, and F259Y. These results indicate that AT1 residues Val254, His256, and Phe259 are not involved in ligand binding but participate in signal transduction. Based in these results and in others from the literature, it is suggested that, in addition to the His256 imidazole ring, the Phe259 aromatic ring interacts with the AII's Phe8, thus contributing to the signal-triggering mechanism.     

Nerve compression syndromes of the extremities

An analogue of human melanin-concentrating hormone (MCH) suitable for radioiodination was designed in which Tyr13 and Val19 of the natural peptide were replaced by phenylalanyl and tyrosyl residues: [Phe13, Tyr19]-MCH. The peptide was synthesized by the continuous-flow solid-phase methodology using Fmoc-strategy and polyhipe PA 500 and PEG-PS resins. The linear MCH peptides with either acetamidomethyl-protected or free cysteinyl residues were purified to homogeneity and cyclized by iodine oxidation, yielding the final product with the correct molecular weight of 2434.61. Radioiodination of the C-terminal tyrosine was carried out enzymatically using solid-phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed-phase mini-column and by high-pressure liquid chromatography. The resulting [125I]-[Phe13, Tyr19]-MCH tracer was the first radiolabelled MCH peptide suitable for radioreceptor assay: saturation binding analysis using mouse G4F-7 melanoma cells demonstrated the presence of 1090 MCH receptors per cell. The dissociation constant (KD) was 1.18 x 10(-10) M, indicating high-affinity MCH receptors on these cells. MCH receptors were also found in other cell lines such as mouse B16-F1 and G4F and human RE melanoma cells as well as in PC12 and COS-7 cells. Competition binding analyses with a number of other peptides such as alpha-MSH, neuropeptide Y, substance P and pituitary adenylate cyclase activating peptide, demonstrated that the binding to the MCH receptor is specific. Atrial natriuretic factor was found to be a weak competitor of MCH, indicating topological similarities between MCH and ANF when interacting with MCH receptors.     

The lipid binding activity of the exchangeable apolipoprotein apolipophorin-III correlates with the formation of a partially folded conformation

Manduca sexta apolipophorin-III, apoLp-III, is an exchangeable apolipoprotein of 17 kDa that contains no Trp, one Tyr, and eight Phe. The effect of pH on the kinetics of association of apoLp-III with dimyristoylphosphatidylcholine was studied. The pH dependence of the kinetics showed three distinct regions. Above pH 7, the reaction rate is slow and slightly affected by pH. A approximately 40-fold increase in the rate constant is observed when the pH is decreased from 8 to 4, and a decrease in rate is observed below pH 4. Far-UV CD spectroscopy indicated that the secondary structure of the protein is not affected when decreasing the pH from 8 to 4.5. The pH dependence of the Tyr fluorescence showed three pH regions that resemble the regions observed in the kinetics. Comparison of the far-UV CD and fluorescence studies indicated the formation of a folding intermediate between pHs 4 and 7. This intermediate was also characterized by near-UV CD and fluorescence quenching. Fluorescence quenching studies with I- and Cs+ indicated a very low exposure of the Tyr residue in both native and intermediate conformations. The pH dependence of the near-UV CD spectra indicated that the native --> intermediate transition is accompanied by a loss in the packing constrains of the Tyr residue. UV absorption spectroscopy of the Phe and Tyr residues indicated that the native --> intermediate transition is also accompanied by the hydration of the Tyr residue and approximately 4 Phe residues. This report shows, for the first time, the correlation between the increase in lipid binding activity of an exchangeable apolipoprotein and the formation of a compact but hydrated conformation near physiological conditions. These results suggest a direct correlation between the lipid binding activity and the internal hydration of the apolipoprotein. The similarity between the insect exchangeable apolipoprotein and the human counterparts, apoA-I, apoA-II, etc., and the recent demonstration of the presence of a molten globular like-state of human apoA-I near physiological conditions [Gursky, O., and Atkinson, D. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 2991-2995] suggest that this highly hydrated and compact state may play an important physiological role as the most active lipid binding state of the apolipoproteins in general.     

Conformational analysis of two cyclic analogs of angiotensin: implications for the biologically active conformation

Conformations of two cyclic analogs of angiotensin (Asp1-Arg2-Val3-Tyr4-Val/Ile5-His6-Pro7-Phe8, AT), cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT, were studied, independently employing two complementary techniques, energy calculations and NMR measurements in DMSO solution. NMR data were indicative of well-defined solution conformations for the cyclic moieties of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT, including the phi values for the Cys3/HCys3 and Tyr4 residues, as well as the chi 1 value for the Tyr4 residue. Solution conformations for the exocyclic linear parts of both molecules cannot be described by the NMR data with the same precision. At the same time, independent energy calculations revealed the same conformations of cyclic moieties of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT among low-energy conformers for both peptides. Moreover, the same conformations are compatible with the model of AT receptor-bound conformation (Nikiforovich & Marshall, 1993), which assumes the particular spatial arrangement of aromatic moieties of Tyr4, His6, and Phe8 residues and the C-terminal carboxyl. These conformers of cyclo[Sar1, Cys3, Mpt5]-AT and cyclo[Sar1, HCys3, Mpt5]-AT contain "an open turn" in the backbone of the Tyr4-Val5 residues, instead of the earlier proposed beta-like reversal, thus confirming the suggestion that the conformation(s) ensuring binding of AT analogs with specific receptors should not be described in terms of a unique backbone conformer.     

Evidence for coupled electron and proton transfer in the [8Fe-7S] cluster of nitrogenase

Substrate reduction by nitrogenase requires electron transfer from a [4Fe-4S] cluster in the iron (Fe) protein component to an FeMo cofactor in the molybdenum-iron (MoFe) protein component in a reaction that is coupled to MgATP hydrolysis and component protein association and dissociation. An [8Fe-7S] (or P-) cluster in the MoFe protein has been proposed as an intermediate electron-transfer site, although how this cluster functions in electron-transfer remains unclear. In the present work, it is demonstrated that one redox couple of the P-cluster (P2+/1+) undergoes coupled electron and proton transfer, whereas a more reduced couple (P1+/N) does not involve a coupled proton transfer. Redox titrations of the MoFe protein P-cluster were performed, and the midpoint potential of the P2+/1+ couple (Em2) was found to be pH dependent, ranging from -224 mV at pH 6.0 to -348 mV at pH 8.5. A plot of Em2 versus the pH for this redox couple was linear and revealed a change of -53 mV/pH unit, indicating a single protonation event associated with reduction. From this plot, it was concluded that p is <6.0 and p is >8.5 in a proton-modified Nernst equation. In contrast, the midpoint potential for the P1+/N couple (Em1) was found to be -290 mV and was invariant over the pH range 6.0-8.5. These results indicate that the protonated species does not influence either the P1+ or the PN oxidation states. In addition, at physiological pH values, electron transfer is coupled to proton transfer for the P2+/1+ couple. The P-clusters are unique among [Fe-S] clusters in that they appear to be ligated to the protein through a serinate-gammaO ligand (betaSer188) and a peptide bond amide-N ligand (alphaCys88), in addition to cysteinate-S ligands. Elimination of the serinate ligand by replacement with a glycine was found to shift the Em values for both P-cluster couples by greater than +60 mV, however the pH dependence of Em2 was unchanged. These results rule out Ser188 as the protonated ligand responsible for the pH dependence of Em2. The implications of these results in understanding the nitrogenase electron-transfer mechanism are discussed.     

Effects of mutations in plastocyanin on the kinetics of the protein rearrangement gating the electron-transfer reaction with zinc cytochrome c. Analysis of the rearrangement pathway

We study, by flash kinetic spectrophotometry on the microsecond time scale, the effects of ionic strength and viscosity on the kinetics of oxidative quenching of the triplet state of zinc cytochrome c (3Zncyt) by the wild-type form and the following nine mutants of cupriplastocyanin: Leu12Glu, Leu12Asn, Phe35Tyr, Gln88Glu, Tyr83Phe, Tyr83His, Asp42Asn, Glu43Asn, and the double mutant Glu59Lys/Glu60Gln. The unimolecular rate constants for the quenching reactions within the persistent diprotein complex, which predominates at low ionic strengths, and within the transient diprotein complex, which is involved at higher ionic strengths, are equal irrespective of the mutation. Evidently, the two complexes are the same. In both reactions, the rate-limiting step is rearrangement of the diprotein complex from a configuration optimal for docking to the one optimal for the subsequent electron-transfer step, which is fast. We investigate the effects of plastocyanin mutations on this rearrangement, which gates the overall electron-transfer reaction. Conversion of the carboxylate anions into amide groups in the lower acidic cluster (residues 42 and 43), replacement of Tyr83 with other aromatic residues, and mutations in the hydrophobic patch in plastocyanin do not significantly affect the rearrangement. Conversion of a pair of carboxylate anions into a cationic and a neutral residue in the upper acidic cluster (residues 59 and 60) impedes the rearrangement. Creation of an anion at position 88, between the upper acidic cluster and the hydrophobic patch, facilitates the rearrangement. The rate constant for the rearrangement smoothly decreases as the solution viscosity increases, irrespective of the mutation. Fittings of this dependence to the modified Kramers's equation and to an empirical equation show that zinc cytochrome c follows the same trajectory on the surfaces of all the plastocyanin mutants but that the obstacles along the way vary as mutations alter the electrostatic potential. Mutations that affect protein association (i.e., change the binding constant) do not necessarily affect the reaction between the associated proteins (i.e., the rate constant) and vice versa. All of the kinetic and thermodynamic effects and noneffects of mutations consistently indicate that in the protein rearrangement the basic patch of zinc cytochrome c moves from a position between the two acidic clusters to a position at or near the upper acidic cluster.     

The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates

The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.     

Importance of extracellular domains for ligand binding in the thyrotropin-releasing hormone receptor

The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor.     

Spectroscopic characterization of a novel tetranuclear Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2. Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.