Azotobacter vinelandii ferredoxin I: a sequence and structure comparison approach to alteration of [4Fe-4S]2+/+ reduction potential.

The reduction potential (E(0)') of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (AvFdI) and related ferredoxins is approximately 200 mV more negative than the corresponding clusters of Peptostreptococcus asaccharolyticus ferredoxin and related ferredoxins. Previous studies have shown that these differences in E(0)' do not result from the presence or absence of negatively charged surface residues or in differences in the types of hydrophobic residues found close to the [4Fe-4S](2+/+) clusters. Recently, a third, quite distinct class of ferredoxins (represented by the structurally characterized Chromatium vinosum ferredoxin) was shown to have a [4Fe-4S](2+/+) cluster with a very negative E(0)' similar to that of AvFdI. The observation that the sequences and structures surrounding the very negative E(0)' clusters in quite dissimilar proteins were almost identical inspired the construction of three additional mutations in the region of the [4Fe-4S](2+/+) cluster of AvFdI. The three mutations, V19E, P47S, and L44S, that incorporated residues found in the higher E(0)' P. asaccharolyticus ferredoxin all led to increases in E(0)' for a total of 130 mV with a 94-mV increase in the case of L44S. The results are interpreted in terms of x-ray structures of the FdI variants and show that the major determinant for the large increase in L44S is the introduction of an OH-S bond between the introduced Ser side chain and the Sgamma atom of Cys ligand 42 and an accompanying movement of water.     

Alteration of the reduction potential of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I

The [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E(0')) relative to other structurally similar ferredoxins. Previous attempts to raise that E(0') by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter the E(0') by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E(0') measurements show that all had altered E(0') relative to native FdI. For P21G FdI and I40Q FdI, the E(0') increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control of E(0'). Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E(0') while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E(0') while a -33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 A, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E(0'). Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E(0').     

Distinct redox behaviour of prosthetic groups in ready and unready hydrogenase from Chromatium vinosum.

The redox behaviour of the Ni(III)/Ni(II) transition in hydrogenase from Chromatium vinosum is described and compared with the redox behaviour of the nickel ion in the F420-nonreducing hydrogenase from Methanobacterium thermoautotrophicum. Analogous to the situation in the oxidised hydrogenase of Desulfovibrio gigas (Fernandez, V.M., Hatchikian, E.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154), the C. vinosum enzyme can also exist in two forms: the 'unready' form (EPR characteristics of Ni(III): gx,y,z = 2.32, 2.24, 2.01) and the 'ready' form (EPR characteristics Ni(III): gx,y,z = 2.34, 2.16, 2.01). Like in the oxidised enzyme of M. thermoautotrophicum the Ni(III)/Ni(II) transition for the unready form titrated completely reversible (both at pH 6.0 and pH 8.0). In contrast, the reversibility of the Ni(III)/Ni(II) transition in the ready enzyme was strongly dependent on pH and temperature. At pH 6.0 and 2 degrees C reduction of Ni(III) in ready enzyme was completely irreversible, whereas at pH 8.0 and 30 degrees C Ni(III) in both ready and unready enzyme titrated with E0' = -115 mV (n = 1). Hampered redox equilibration between the ready enzyme and the mediating dyes is interpreted in terms of an obstruction of the electron transfer from nickel at the active site to the artificial electron acceptors in solution. The origin of this obstruction might be related to possible changes in the protein structure induced by the activation process. The E0'-value of the Ni(III)/Ni(II) equilibrium was pH sensitive (-60 mV/delta pH) indicating that reduction of nickel is coupled to a protonation. A similar pH-dependence was observed for the titration of the spin-spin interaction of Ni(III) and a special form of the [3Fe-4S]+ cluster (E0' = +150 mV, pH 8.0, 30 degrees C). Redox equilibration of this coupling was extremely sensitive to pH and temperature. The uncoupled [3Fe-4S]+ cluster titrated pH-independently with E0' = -10 mV (pH 8.0, 30 degrees C).     

Effects of noncovalent and covalent FAD binding on the redox and catalytic properties of p-cresol methylhydroxylase

Each flavoprotein subunit (alpha or PchF) of the alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida contains FAD covalently attached to Tyr384. PCMH oxidizes p-cresol to 4-hydroxybenzyl alcohol, which is oxidized subsequently by PCMH to 4-hydroxybenzaldehyde. The Y384F mutant form of PchF (apo-PchF[Y384F]) displayed stoichiometric noncovalent FAD binding. PchF[Y384F]FAD associated with the cytochrome subunit (beta or PchC) (producing PCMH[Y384F]), although not as avidly as with wild-type PchF containing covalently bound FAD (PchF(C)). Dramatic increases in the two-electron E(m,7) (NHE) values for FAD were observed when it bound noncovalently to either apo-PchF or apo-PchF[Y384F], and the two-electron E(m,7) value for FAD was increased further by about 75 mV upon covalent binding to PchF, i.e., PchF(C). The E(m,7) values increased by approximately 20 and 45 mV, respectively, when PchF(C) and PchF[Y384F]FAD associated with PchC. The two-electron E(m,7) for covalently bound FAD in PCMH is 84 mV, the highest measured for a flavoprotein. The values for the one-electron redox potentials (E(m,7), NHE) for FAD were measured also for various forms of PchF. Under anaerobiosis, the reduction of PchF[Y384F]FAD by substrates was similar to that observed previously for PchF containing noncovalently bound FAD. Stopped-flow kinetic studies indicated a rapid substrate reduction of the FAD and heme in PCMH[Y384F] which produced PchF[Y384F]FAD(rad) x PchC, the mutant enzyme containing the flavin radical and reduced heme. These experiments also revealed a slow reduction of unassociated PchC(ox) by PchF[Y384F]FAD(rad) x PchC. Steady-state kinetic studies of the reaction of PCMH[Y384F] with p-cresol indicated that the K(m) for this substrate was unchanged relative to that of PCMH, but that the k(cat) was diminished by an order of magnitude. The data indicate that the covalent attachment of FAD to PchF assists catalysis by raising the E(m,7) of the flavin. Contributions to this effect likely result from conformational changes.     

Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy

Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe-4S]1+, [4Fe-4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA-SerB-SerC, a hetero-trimetric complex of alphabetagamma subunits) revealed that the [3Fe-4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (E(m)) of +118+/-10 mV for the [3Fe-4S]1+/0 couple. A [4Fe-4S]1+ cluster EPR signal developed over a range of potentials between 300 and -200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183+/-10 mV (FS1) and -51+/-10 mV (FS3) for the two [4Fe-4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe-4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (E(m)) of about -356 mV (FS2). It is considered likely that, at low redox potential (E(m) less than -300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe-4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe-4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3 approximately 1.999, g2 approximately 1.996 and g1 approximately 1.965 (g(av) 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g approximately 3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.     

Electron transfer from heme bL to the [3Fe-4S] cluster of Escherichia coli nitrate reductase A (NarGHI)

We have investigated the functional relationship between three of the prosthetic groups of Escherichia coli nitrate reductase A (NarGHI): the two hemes of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster of the electron-transfer subunit (NarH). In two site-directed mutants (NarGHI(H56R) and NarGHI(H205Y)) that lack the highest potential heme of NarI (heme b(H)), a large negative DeltaE(m,7) is elicited on the NarH [3Fe-4S] cluster, suggesting a close juxtaposition of these two centers in the holoenzyme. In a mutant retaining heme b(H), but lacking heme b(L) (NarGHI(H66Y)), there is no effect on the NarH [3Fe-4S] cluster redox properties. These results suggest a role for heme b(H) in electron transfer to the [3Fe-4S] cluster. Studies of the pH dependence of the [3Fe-4S] cluster, heme b(H), and heme b(L) E(m) values suggest that significant deprotonation is only observed during oxidation of the latter heme (a pH dependence of -36 mV pH(-1)). In NarI expressed in the absence of NarGH [NarI(DeltaGH)], apparent exposure of heme b(H) to the aqueous milieu results in both it and heme b(L) having E(m) values with pH dependencies of approximately -30 mV pH(-1). These results are consistent with heme b(H) being isolated from the aqueous milieu and pH effects in the holoenzyme. Optical spectroscopy indicates that inhibitors such as HOQNO and stigmatellin bind and inhibit oxidation of heme b(L) but do not inhibit oxidation of heme b(H). Fluorescence quench titrations indicate that HOQNO binds with higher affinity to the reduced form of NarGHI than to the oxidized form. Overall, the data support the following model for electron transfer through the NarI region of NarGHI: Q(P) site --> heme b(L) --> heme b(H) --> [3Fe-4S] cluster.     

Potentiometric and electron nuclear double resonance properties of the two spin forms of the [4Fe-4S]+ cluster in the novel ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus.

Pyrococcus furiosus ferredoxin contains a single [4Fe-4S] that exists in both S = 1/2 (20%) and S = 3/2 (80%) ground states in the reduced protein. We report here on the temperature-dependent potentiometric properties of the two spin forms, their stability, and on the structural features that differentiate them. The midpoint potential (Em) of the cluster in either spin state was determined at -365 mV (30 degrees C, pH 8.0). By rapidly freezing samples for EPR analyses, it was shown that the Em values of both spin states appear to change by -1.7 mV/degrees C over the range 20 degrees-80 degrees C, and by -6 mV/degrees C between 80 and 89 degrees C. The Em values and the relative amounts of the S = 1/2 and S = 3/2 forms of the cluster were unaffected by pH (6.8-10.5), even at 85 degrees C, and were unchanged by the presence of NaCl (1.0 M), sodium dodecyl sulfate (10%, w/v) or ethylene glycol (50%, v/v), even at 80 degrees C. The S = 1/2 form of the [4Fe-4S]+ cluster was found to exhibit a strongly coupled 1H ENDOR resonance (A = 22 MHz) that was exchangeable with the solvent. Such a large coupling has not been observed in any other iron-sulfur protein. Since a unique feature of this 4Fe-ferredoxin is that only 3 cysteinyl residues appear to be coordinated to the [4Fe-4S] cluster, the ENDOR data are consistent with an H2O molecule being a ligand to the unique Fe site. The S = 3/2 form of the [4Fe-4S]+ cluster exhibited a similar, strongly coupled 1H ENDOR resonance, but in this spin state it was not exchangeable with the solvent. This suggests that the [4Fe-4S]+ cluster exhibiting the S = 3/2, but not the S = 1/2 ground state, is "shielded" from the solvent, presumably by neighboring amino acid residues. In view of the pH dependence of the midpoint potential of the two spin states, the fourth ligand to the cluster and the source of the strongly coupled 1H ENDOR resonance is probably an OH- rather than H2O molecule.     

Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus

We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.     

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.     

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I. Changes in [4Fe-4S] cluster reduction potential and reactivity.

We have used site-directed mutagenesis to obtain two variants of Azotobacter vinelandii ferredoxin I (AvFdI), whose x-ray structures are now available. In the C20A protein, a ligand to the [4Fe-4S] cluster was removed whereas in the C24A mutant a free cysteine next to that cluster was removed. Like native FdI, both mutants contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. The structure of C24A is very similar to that of native FdI, while the structure of C20A is rearranged in the region of the [4Fe-4S] cluster to allow it to use the free Cys-24 as a replacement ligand. Here we compare the properties of the native, C20A, and C24A proteins. Although all three proteins are O2 stable in vitro, the C20A protein is much less stable toward proteolysis than the other two in vivo. Spectroscopic results show that all three proteins exhibit the same general redox behavior during O2-oxidation and dithionite reduction. Electrochemical data show that the [3Fe-4S] clusters in all three proteins have the same pH-dependent reduction potentials (-425 mV versus SHE, pH 7.8), whereas the [4Fe-4S] cluster potentials vary over a approximately 150 mV range from -600 mV (C24A) to -647 mV (native) to -746 mV (C20A). Despite this variation in potential both the C20A and C24A proteins appear to be functional in vivo. Native FdI reacts with three equivalents of Fe(CN)3-(6) to form a paramagnetic species previously proposed to be a cysteinyl-disulfide radical. Neither the C20A nor the C24A variant undergoes this reaction, strongly suggesting that it involves the free Cys-24.     

Characterization of benzodiazepine binding site on human α1-acid glycoprotein using flunitrazepam as a photolabeling agent

The binding of flunitrazepam (FNZP) by human α₁-acid glycoprotein (hAGP) and the relationships between the extent of drug binding and desialylation and the genetic variants of hAGP were examined. The photolabeling specificity of [³H]FNZP was confirmed by findings in which other hAGP-binding ligands inhibited the formation of covalent bonds between [³H]FNZP and hAGP. The photolabeling of asialo-hAGP suggested that sialic acid does not involve in the binding of [³H]FNZP. No difference in the labeling could be found between the F1 * S variants and A variant. Similarly, FNZP did not show a difference in binding affinity to the two genetic variants of hAGP. Sequence analysis of the photolabeled peptide indicated a sequence corresponding to Tyr91-Arg105 of hAGP.     

Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via site-directed mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster

The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine-2'-deoxyadenosine (OG*A) mismatches in DNA. MutY prevents DNA mutations caused by the misincorporation of A opposite OG by catalyzing the deglycosylation of the aberrant adenine. MutY is representative of a unique subfamily of DNA repair enzymes that also contain a [4Fe-4S]2+ cluster, which has been implicated in substrate recognition. Previously, we have used site-directed mutagenesis to individually replace the cysteine ligands to the [4Fe-4S]2+ cluster of E. coli MutY with serine, histidine, or alanine. These experiments suggested that histidine coordination to the iron-sulfur cluster may be accommodated in MutY at position 199. Purification and enzymatic analysis of C199H and C199S forms indicated that these forms behave nearly identical to the WT enzyme. Furthermore, introduction of the C199H mutation in a truncated form of MutY (C199HT) allowed for crystallization and structural characterization of the modified [4Fe-4S] cluster coordination. The C199HT structure showed that histidine coordinated to the iron cluster although comparison to the structure of the WT truncated enzyme indicated that the occupancy of iron at the modified position had been reduced to 60%. Electron paramagnetic resonance (EPR) spectroscopy on samples of C199HT indicates that a significant percentage (15-30%) of iron clusters were of the [3Fe-4S]1+ form. Oxidation of the C199HT enzyme with ferricyanide increases the amount of the 3Fe cluster by approximately 2-fold. Detailed kinetic analysis on samples containing a mixture of [3Fe-4S]1+ and [4Fe-4S]2+ forms indicated that the reactivity of the [3Fe-4S]1+ C199HT enzyme does not differ significantly from that of the WT truncated enzyme. The relative resistance of the [4Fe-4S]2+ cluster toward oxidation, as well as the retention of activity of the [3Fe-4S]1+ form, may be an important aspect of the role of MutY in repair of DNA damage resulting from oxidative stress.     

Hydrophobic modulation of heme properties in heme protein maquettes

We have investigated the properties of the two hemes bound to histidine in the H10 positions of the uniquely structured apo form of the heme binding four-helix bundle protein maquette [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2) for the amino acids at positions 6 (I), 13 (F) and 24 (H), respectively. The primary structure of each alpha-helix, alpha-SH, in [I(6)F(13)H(24)](2) is Ac-CGGGEI(6)WKL.H(10)EEF(13)LKK.FEELLKL.H(24)EERLKK.L-CONH(2). In our nomenclature, [I(6)F(13)H(24)] represents the disulfide-bridged di-alpha-helical homodimer of this sequence, i.e., (alpha-SS-alpha), and [I(6)F(13)H(24)](2) represents the dimeric four helix bundle composed of two di-alpha-helical subunits, i.e., (alpha-SS-alpha)(2). We replaced the histidines at positions H24 in [I(6)F(13)H(24)](2) with hydrophobic amino acids incompetent for heme ligation. These maquette variants, [I(6)F(13)I(24)](2), [I(6)F(13)A(24)](2), and [I(6)F(13)F(24)](2), are distinguished from the tetraheme binding parent peptide, [I(6)F(13)H(24)](2), by a reduction in the heme:four-helix bundle stoichiometry from 4:1 to 2:1. Iterative redesign has identified phenylalanine as the optimal amino acid replacement for H24 in the context of apo state conformational specificity. Furthermore, the novel second generation diheme [I(6)F(13)F(24)](2) maquette was related to the first generation diheme [H10A24](2) prototype, [L(6)L(13)A(24)](2) in the present nomenclature, via a sequential path in sequence space to evaluate the effects of conservative hydrophobic amino acid changes on heme properties. Each of the disulfide-linked dipeptides studied was highly helical (>77% as determined from circular dichroism spectroscopy), self-associates in solution to form a dimer (as determined by size exclusion chromatography), is thermodynamically stable (-DeltaG(H)2(O) >18 kcal/mol), and possesses conformational specificity that NMR data indicate can vary from multistructured to single structured. Each peptide binds one heme with a dissociation constant, K(d1) value, tighter than 65 nM forming a series of monoheme maquettes. Addition of a second equivalent of heme results in heme binding with a K(d2) in the range of 35-800 nM forming the diheme maquette state. Single conservative amino acid changes between peptide sequences are responsible for up to 10-fold changes in K(d) values. The equilibrium reduction midpoint potential (E(m7.5)) determined in the monoheme state ranges from -156 to -210 mV vs SHE and in the diheme state ranges from -144 to -288 mV. An observed heme-heme electrostatic interaction (>70 mV) in the diheme state indicates a syn global topology of the di-alpha-helical monomers. The heme affinity and electrochemistry of the three H24 variants studied identify the tight binding sites (K(d1) and K(d2) values <200 nM) having the lower reduction midpoint potentials (E(m7.5) values of -155 and -260 mV) with the H10 bound hemes in the parent tetraheme state of [H10H24-L6I,L13F](2), here called [I(6)F(13)H(24)](2). The results of this study illustrate that conservative hydrophobic amino acid changes near the heme binding site can modulate the E(m) by up to +/-50 mV and the K(d) by an order of magnitude. Furthermore, the effects of multiple single amino acid changes on E(m) and K(d) do not appear to be additive.     

Self-assembly of heme A and heme B in a designed four-helix bundle: implications for a cytochrome c oxidase maquette

Heme A, a prosthetic group of cytochrome c oxidase [EC 1.9.3.1], has been introduced into two de novo designed four helix bundle proteins, [H10A24](2) and [H10H24](2), known to bind 2-4 equiv of heme B, respectively [Robertson, D. E., Farid, R. S., Moser, C. C., Mulholland, S. E., Pidikiti, R., Lear, J. D., Wand, A., J., DeGrado, W. F., and Dutton, P. L. (1994) Nature 368, 425-432]. [H10A24](2), [Ac-CGGGELWKL x HEELLKK x FEELLKL x AEERLKK x L-CONH(2)](2)(2), binds two heme A molecules per four-helix unit via bis-histidine ligation at the 10,10' positions with measured K(d) values of <0.1 and 5 nM, values much lower than those measured for heme B (K(d) values of 50 and 800 nM). The heme A-protein complex, [heme A-H10A24](2), exhibits well-defined absorption spectra in both the ferric and ferrous states, and an electron paramagnetic resonance spectrum characteristic of a low spin heme in the ferric form. A single midpoint redox potential (E(m8)) was determined for [heme A-H10A24](2) at -45 mV (vs SHE), which is significantly higher than that of the protein bound heme B (-130 and -200 mV). The observation of a single midpoint redox potential for [heme A-H10A24](2) and a pair of midpoints for [heme B-H10A24](2) indicates that the di-alpha-helical monomers are oriented in an anti topology (disulfides on opposite sides of bundle) in the former (lacking heme-heme electrostatic interaction) and syn in the latter. A mixture of global topologies was indicated by the potentiometric titration of the related [heme A-H10H24](2) which possess two distinct reduction potentials of +41 (31%) and -65 mV (69%). Self-assembly of the mixed cofactor heme A-heme B-[H10A24](2) was accomplished by addition of a single equivalent of each heme A and heme B to [H10A24](2). The single midpoint redox potential of heme B, E(m8) = -200 mV, together with the split midpoint redox potential of heme A in heme A-heme B-[H10A24](2), E(m8) = +28 mV (33%) and -65 mV (67%), indicated the existence of both syn and anti topologies of the two di-alpha-helical monomers in this four helix bundle. Synthesis of the mixed cofactor [heme A-heme B-H10H24](2) was accomplished by addition of a 2 equiv of each heme A and heme B to [H10H24](2) and potentiometry indicated the pair of hemes B resided in the 10,10' sites and heme A occupied the 24,24' sites. The results indicate that heme peripheral structure controls the orientation of the di-alpha-helical monomers in the four-helix bundle which are interchangeable between syn and anti topologies. In the reduced form, [heme A-H10A24](2), reacts quantitatively to form [carbonmonoxy-heme A-H10A24](2) as evidenced by optical spectroscopy. The synthetic [heme A-H10A24](2) can be enzymatically reduced by NAD(P)H with natural reductases under anaerobic conditions, and reversibly oxidized by dioxygen to the ferric form.     

Electrochemical and spectroscopic characterization of the 7Fe form of ferredoxin III from Desulfovibrio africanus.

Desulfovibrio africanus ferredoxin III is a monomeric protein (Mr 6585) containing seven cysteine residues and 7-8 iron atoms and 6-8 atoms of acid-labile sulphur. It is shown that reversible unmediated electrochemistry of the two iron-sulphur clusters can be obtained by using a pyrolytic-graphite-'edge' carbon electrode in the presence of an appropriate aminoglycoside, neomycin or tobramycin, as promoter. Cyclic voltammetry reveals two well-defined reversible waves with E0' = -140 +/- 10 mV and -410 +/- 5 mV (standard hydrogen electrode) at 2 degrees C. Bulk reduction confirms that each of these corresponds to a one-electron process. Low-temperature e.p.r. and magnetic-c.d. spectroscopy identify the higher-potential redox couple with a cluster of core [3Fe-4S]1+.0 and the lower with a [4Fe-4S]2+.1+ centre. The low-temperature magnetic-c.d. spectra and magnetization properties of the three-iron cluster show that it is essentially identical with that in Desulfovibrio gigas ferredoxin II. We assign cysteine-11, -17 and -51 as ligands of the [3Fe-4S] core and cysteine-21, -41, -44 and -47 to the [4Fe-4S] centre.     

Effect of active site and surface mutations on the reduction potential of yeast cytochrome c peroxidase and spectroscopic properties of the oxidized and reduced enzyme

The reduction potentials of 22 yeast cytochrome c peroxidase (CcP) mutants were determined at pH 7.0 in order to determine the effect of both heme pocket and surface mutations on the Fe(III)/Fe(II) redox couple of CcP, as well as to determine the range in redox potentials that could be obtained through point mutations in the enzyme. Spectroscopic properties of the Fe(III) and Fe(II) forms of the mutant enzymes are also reported. The mutations include variants in the distal and proximal heme pockets as well as on the enzyme surface and involve single, double, and triple point mutations. A spectrochemical redox titration technique used in this study gave an E(0') value of -189 mV for yeast CcP compared to a previously reported value of -194 mV determined by potentiometry [C.W. Conroy, P. Tyma, P.H. Daum, J.E. Erman, Biochim. Biophys. Acta 537 (1978) 62-69]. Both positive and negative shifts in the reduction potential from that of the wild-type enzyme were observed, spanning a range of 113 mV. The His-52-->Asn mutation gave the most negative potential, -259 mV, while a triple mutant in which the three distal pocket residues, Arg-48, Trp-51, and His-52, were all converted to leucine residues gave the most positive potential, -146 mV.     

The interaction of 5'-adenylylsulfate reductase from Pseudomonas aeruginosa with its substrates

APS reductase from Pseudomonas aeruginosa has been shown to form a disulfide-linked adduct with mono-cysteine variants of Escherichia coli thioredoxin and Chlamydomonas reinhardtii thioredoxin h1. These adducts presumably represent trapped versions of the intermediates formed during the catalytic cycle of this thioredoxin-dependent enzyme. The oxidation-reduction midpoint potential of the disulfide bond in the P. aeruginosa APS reductase/C. reinhardtii thioredoxin h1 adduct is -280 mV. Site-directed mutagenesis and mass spectrometry have identified Cys256 as the P. aeruginosa APS reductase residue that forms a disulfide bond with Cys36 of C. reinhardtii TRX h1 and Cys32 of E. coli thioredoxin in these adducts. Spectral perturbation measurements indicate that P. aeruginosa APS reductase can also form a non-covalent complex with E. coli thioredoxin and with C. reinhardtii thioredoxin h1. Perturbation of the resonance Raman and visible-region absorbance spectra of the APS reductase [4Fe-4S] center by either APS or the competitive inhibitor 5'-AMP indicates that both the substrate and product bind in close proximity to the cluster. These results have been interpreted in terms of a scheme in which one of the redox-active cysteine residues serves as the initial reductant for APS bound at or in close proximity to the [4Fe-4S] cluster.     

The [2Fe-2S] cluster E(m) as an indicator of the iron-sulfur subunit position in the ubihydroquinone oxidation site of the cytochrome bc1 complex.

Recent crystallographic and kinetic data have revealed the crucial role of the large scale domain movement of the iron-sulfur subunit [2Fe-2S] cluster domain during the ubihydroquinone oxidation reaction catalyzed by the cytochrome bc(1) complex. Previously, the electron paramagnetic resonance signature of the [2Fe-2S] cluster and its redox midpoint potential (E(m)) value have been used extensively to characterize the interactions of the [2Fe-2S] cluster with the occupants of the ubihydroquinone oxidation (Q(o)) catalytic site. In this work we analyze these interactions in various iron-sulfur subunit mutants that carry mutations in its flexible hinge region. We show that the E(m) increases of the iron-sulfur subunit [2Fe-2S] cluster induced either by these mutations or by the addition of stigmatellin do not act synergistically. Moreover, the E(m) increases disappear in the presence of class I inhibitors like myxothiazol. Because various inhibitors are known to affect the location of the iron-sulfur subunit cluster domain, the measured E(m) value of the [2Fe-2S] cluster therefore reflects its equilibrium position in the Q(o) site. We also demonstrate the existence in this site of a location where the E(m) of the cluster is increased by about 150 mV and discuss its possible implications in term of Q(o) site catalysis and energetics.     

A conserved lysine residue controls iron–sulfur cluster redox chemistry in Escherichia coli fumarate reductase

The Escherichia coli respiratory complex II paralogs succinate dehydrogenase (SdhCDAB) and fumarate reductase (FrdABCD) catalyze interconversion of succinate and fumarate coupled to quinone reduction or oxidation, respectively. Based on structural comparison of the two enzymes, equivalent residues at the interface between the highly homologous soluble domains and the divergent membrane anchor domains were targeted for study. This included the residue pair SdhB-R205 and FrdB-S203, as well as the conserved SdhB-K230 and FrdB-K228 pair. The close proximity of these residues to the [3Fe–4S] cluster and the quinone binding pocket provided an excellent opportunity to investigate factors controlling the reduction potential of the [3Fe–4S] cluster, the directionality of electron transfer and catalysis, and the architecture and chemistry of the quinone binding sites. Our results indicate that both SdhB-R205 and SdhB-K230 play important roles in fine tuning the reduction potential of both the [3Fe–4S] cluster and the heme. In FrdABCD, mutation of FrdB-S203 did not alter the reduction potential of the [3Fe–4S] cluster, but removal of the basic residue at FrdB-K228 caused a significant downward shift (> 100 mV) in potential. The latter residue is also indispensable for quinone binding and enzyme activity. The differences observed for the FrdB-K228 and Sdh-K230 variants can be attributed to the different locations of the quinone binding site in the two paralogs. Although this residue is absolutely conserved, they have diverged to achieve different functions in Frd and Sdh.     

Unusual NMR, EPR, and M?ssbauer properties of Chromatium vinosum 2[4Fe-4S] ferredoxin.

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and M?ssbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its M?ssbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.