Redox-dependent structural reorganization in putidaredoxin, a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida

Putidaredoxin (Pdx), a vertebrate-type [2Fe-2S] ferredoxin from Pseudomonas putida, transfers electrons from NADH-putidaredoxin reductase to cytochrome P450cam. Pdx exhibits redox-dependent binding affinities for P450cam and is thought to play an effector role in the monooxygenase reaction catalyzed by this hemoprotein. To understand how the reduced form of Pdx is stabilized and how reduction of the [2Fe-2S] cluster affects molecular properties of the iron-sulfur protein, crystal structures of reduced C73S and C73S/C85S Pdx were solved to 1.45 angstroms and 1.84 angstroms resolution, respectively, and compared to the corresponding 2.0 angstroms and 2.03 angstroms X-ray models of the oxidized mutants. To prevent photoreduction, the latter models were determined using in-house radiation source and the X-ray dose received by Pdx crystals was significantly decreased. Structural analysis showed that in reduced Pdx the Cys45-Ala46 peptide bond flip initiates readjustment of hydrogen bonding interactions between the [2Fe-2S] cluster, the Sgamma atoms of the cysteinyl ligands, and the backbone amide nitrogen atoms that results in tightening of the Cys39-Cys48 metal cluster binding loop around the prosthetic group and shifting of the metal center toward the Cys45-Thr47 peptide. From the metal center binding loop, the redox changes are transmitted to the linked Ile32-Asp38 peptide triggering structural rearrangement between the Tyr33-Asp34, Ser7-Asp9 and Pro102-Asp103 fragments of Pdx. The newly established hydrogen bonding interactions between Ser7, Asp9, Tyr33, Asp34, and Pro102, in turn, not only stabilize the tightened conformation of the [2Fe-2S] cluster binding loop but also assist in formation of a specific structural patch on the surface of Pdx that can be recognized by P450cam. This redox-linked change in surface properties is likely to be responsible for different binding affinity of oxidized and reduced Pdx to the hemoprotein.     

Crystal structure of Escherichia coli Fdx, an adrenodoxin-type ferredoxin involved in the assembly of iron-sulfur clusters

Escherichia coli ferredoxin (Fdx) is an adrenodoxin-type [2Fe-2S] ferredoxin. Recent genetic analyses show that it has an essential role in the maturation of various iron-sulfur (Fe-S) proteins. Fdx probably functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. Its crystal structure was determined by the multiple-wavelength anomalous dispersion method using the iron atoms in the [2Fe-2S] cluster of the protein and then refined to R and R(free) values of 0.255 and 0.278, respectively, at 1.7 A resolution. The structure of Fdx is similar to the structures of bovine adrenodoxin (Adx) and Pseudomonas putida putidaredoxin (Pdx) whose respective root-mean-square deviations of the corresponding Calpha atoms are 1.8 and 2.2 A. This analysis also revealed the structure of the C-terminal residues protruding into the solvent, which is missing in Adx and Pdx. The [2Fe-2S] cluster is located at the edge of the molecule and bonds with the Sgamma atoms of Cys42, Cys48, Cys51, and Cys87. Electrostatic potential analysis showed that the surface of Fdx has two negatively charged areas separated by a hydrophobic lane. One is conserved on the surface of Adx which is an area of interaction with adrenodoxin reductase. Cys46 is located on the molecular surface in the vicinity of the [2Fe-2S] cluster, an indication that it may be involved in Fe-S cluster formation.     

Putidaredoxin-to-cytochrome P450cam electron transfer: differences between the two reductive steps required for catalysis

Cytochrome P450cam (P450cam) is the terminal monooxygenase in a three-component camphor-hydroxylating system from Pseudomonas putida. The reaction cycle requires two distinct electron transfer (ET) processes from the [2Fe-2S] containing putidaredoxin (Pdx) to P450cam. Even though the mechanism of interaction and ET between the two proteins has been under investigation for over 30 years, the second reductive step and the effector role of Pdx are not fully understood. We utilized mutagenesis, kinetic, and computer modeling approaches to better understand differences between the two Pdx-to-P450cam ET events. Our results indicate that interacting residues and the ET pathways in the complexes formed between reduced Pdx (Pdx(r)) and the ferric and ferrous dioxygen-bound forms of P450cam (oxy-P450cam) are different. Pdx Asp38 and Trp106 were found to be key players in both reductive steps. Compared to the wild-type Pdx, the D38A, W106A, and delta106 mutants exhibited considerably higher Kd values for ferric P450cam and retained ca. 20% of the first electron transferring ability. In contrast, the binding affinity of the mutants for oxy-P450cam was not substantially altered while the second ET rates were <1%. On the basis of the kinetic and modeling data we conclude that (i) P450cam-Pdx interaction is highly specific in part because it is guided/controlled by the redox state of both partners; (ii) there are alternative ET routes from Pdx(r) to ferric P450cam and a unique pathway to oxy-P450cam involving Asp38; (iii) Pdx Trp106 is a key structural element that couples the second ET event to product formation possibly via its "push" effect on the heme-binding loop.     

The putidaredoxin reductase-putidaredoxin electron transfer complex: theoretical and experimental studies

Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from Pseudomonas putida was studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 A. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp(38) and Trp(106), and at the edge of the protein-protein interface, Tyr(33) and Arg(66), were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Delta106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Delta106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp(106) and Tyr(33)/Arg(66) substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr(33), Arg(66), and Trp(106) prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp(106) but are likely to include Asp(38) and Cys(39).     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.     

Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis.

We have used site-directed mutagenesis to alter the [Fe-S] cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC). The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 [4Fe-4S] clusters [Cammack, R., & Weiner, J. H. (1990) Biochemistry 29, 8410-8416]. Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E. coli nitrate reductase (NarGHJI), which contains a [3Fe-4S] cluster in addition to multiple [4Fe-4S] clusters. The Cys group primarily involved in providing ligands to the [3Fe-4S] cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB. We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties. The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO. The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states. In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy). The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the [4Fe-4S] clusters present in the wild-type enzyme into [3Fe-4S] clusters in the mutant enzymes.     

Increasing the Catalytic Performance of a Whole Cell Biocatalyst Harboring a Cytochrome P450cam System by Stabilization of an Electron Transfer Component

Catalytic activity of a recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system from Pseudomonas putida coupled with enzymatic co-factor regeneration was investigated. About 0.7 μmol camphor was hydroxylated per mg dry cells at 4°C in 50 mM Tris/HCl buffer (pH 7.4) when utilizing a stable putidaredoxin (Pdx) mutant, C73S/C85S-Pdx (Cys73Ser, Cys85Ser double mutant), instead of wild-type Pdx, which was about two-fold improvement in the substrate conversion. Ten-micromole camphor was completely hydroxylated at 20°C in 6 h by 15 mg dry cell weight of whole cell biocatalyst including C73S/C85S-Pdx. Thus, modulation of protein-protein interaction in multicomponent enzymatic catalysis in whole cells is important.     

Redox-dependent structural differences in putidaredoxin derived from homologous structure refinement via residual dipolar couplings

Structural differences in the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), from the camphor hydroxylation pathway of Pseudomonas putida have been investigated as a function of oxidation state of the iron cluster. Pdx is involved in biological electron transfer to cytochrome P450(cam) (CYP101). Redox-dependent differences have been observed previously for Pdx in terms of binding affinities to CYP101, NMR spectral differences, and dynamic properties. To further characterize these differences, structure refinement of both oxidized and reduced Pdx has been carried out using a hybrid approach utilizing paramagnetic distance restraints and NMR orientational restraints in the form of backbone (15)N residual dipolar couplings. Use of these new restraints has improved the structure of oxidized Pdx considerably over the earlier solution NMR structure without RDC restraints, with the new structure now much closer in overall fold to the recently published X-ray crystal structures. We now observe better defined relative orientations of the major secondary structure elements as also of the conformation of the metal binding loop region. Extension of this approach to structure calculation of reduced Pdx has identified structural differences that are primarily localized for residues in the C-terminal interaction domain consisting of the functionally important residue Trp 106 and regions near the metal binding loop in Pdx. These redox-dependent structural differences in Pdx correlate to dynamic changes observed before and may be linked to differences in binding and electron transfer properties between oxidized and reduced Pdx.     

Comparison of functional domains in vertebrate-type ferredoxins

The vertebrate-type Cys(4)Fe(2)S(2) ferredoxins are a class of small acidic proteins that typically act as electron shuttles between NAD(P)H-dependent reductases and monoxygenases, particularly cytochromes P450. Nuclear magnetic resonance assignments and detailed analysis of nuclear Overhauser effects permit the direct comparison of the functional C-terminal domains of three vertebrate-type ferredoxins, the mammalian adrenodoxin (Adx) and the bacterial ferredoxins putidaredoxin (Pdx) and terpredoxin (Tdx). In particular, homologous hydrogen-bonding networks involving a conserved basic residue (His 49 in Pdx, His 56 in Adx, Arg 49 in Tdx) are detailed. This hydrogen bond network appears to play a role in the mechanical transmission of redox-dependent conformational and dynamic changes from the iron-sulfur binding loop to the C-terminal domain. Hydrogen/deuterium exchange measurements have been made in Adx as a function of oxidation state for comparison with previous studies of Pdx and Tdx. The results of these measurements highlight the importance of the conserved basic residue in the linkage between oxidation state and protein dynamics. Finally, a series of mutations have been made in the C-terminal domain of Pdx, including one, Y51F, that disrupts the proposed hydrogen-bonding network without perturbing steric and hydrophobic interactions in the functional domain. Although the mutant is considerably destabilized with respect to wild-type Pdx, relatively unperturbed chemical shifts for residues near the site of the mutation and NOEs between water and Phe 51 suggest that the network is reconstituted with a solvent water in place of the tyrosine hydroxyl group in this mutant.     

1H NMR sequential assignments and identification of secondary structural elements in oxidized putidaredoxin, an electron-transfer protein from Pseudomonas.

Sequential 1H resonance assignments and secondary structural features of putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, are reported. No crystal structure has been obtained for Pdx or for any closely homologous protein. The sequentially assigned resonances represent ca. 83% of all the protons in Pdx and a large majority of those protons which are unaffected by the paramagnetism of the iron-sulfur cluster. A total of three alpha-helices, two beta-sheets, and two type I beta-turns have been identified from NOE (nuclear Overhauser effect) patterns. Besides the extensive beta-sheet described previously, a second sheet is identified, consisting of two short antiparallel strands (Ile 89-Thr 91 and Val 21-Leu 23), one of which ends in a tight type I turn (Thr 91-Pro 92-Glu 93-Leu 94). One short helix (Ala 26-Gly 31) and a second longer helical region (Glu 54-Cys 73) are present. This second helical region is discontinuous, breaking at Pro 61, resuming at Glu 65, and ending at Cys 73. The functionally important C-terminal tryptophan residue has been identified, and some structural constraints on this residue are described. Previously reported functional data concerning Pdx are discussed in light of present structural information. Finally, approaches to the determination of a high-resolution solution structure of the protein are discussed.     

A new electron transport mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin

The adrenal ferredoxin (adrenodoxin, Adx) is an acidic 14.4-kDa [2Fe-2S] ferredoxin that belongs to the vertebrate ferredoxin family. It is involved in the electron transfer from the flavoenzyme NADPH-adrenodoxin-reductase to cytochromes P-450(scc) and P-450(11)(beta). The interaction between the redox partners during electron transport has not yet been fully established. Determining the tertiary structure of an electron-transfer protein may be very helpful in understanding the transport mechanism. In the present work, we report a structural study on the oxidized and reduced forms of bovine adrenodoxin (bAdx) in solution using high-resolution NMR spectroscopy. The protein was produced in Escherichia coli and singly or doubly labeled with (15)N or (13)C/(15)N, respectively. Approximately 70 and 75% of the (15)N, (13)C, and (1)H resonances could be assigned for the reduced and the oxidized bAdx, respectively. The secondary and tertiary structures of the reduced and oxidized states were determined using NOE distance information. (1)H(N)-T(1) relaxation times of certain residues were used to obtain additional distance constraints to the [2Fe-2S] cluster. The results suggest that the solution structure of oxidized Adx is quite similar to the X-ray structure. However, structural changes occur upon reduction of the [2Fe-2S] cluster, as indicated by NMR measurements. It could be shown that these conformational changes, especially in the C-terminal region, cause the dissociation of the Adx dimer upon reduction. A new electron transport mechanism proceeding via a modified shuttle mechanism, with both monomers and dimers acting as electron carriers, is proposed.     

Light-induced reduction of bovine adrenodoxin via the covalently bound ruthenium(II) bipyridyl complex: intramolecular electron transfer and crystal structure

Bovine adrenodoxin (Adx) plays an important role in the electron-transfer process in the mitochondrial steroid hydroxylase system of the bovine adrenal cortex. Using electron paramagnetic resonance (EPR) spectroscopy, we showed that photoreduction of the [2Fe-2S] cluster of Adx via (4'-methyl-2,2'-bipyridine)bis(2,2'-bipyridine)ruthenium(II) [Ru(bpy)2(mbpy)] covalently attached to the protein surface can be used as a new approach to probe the "shuttle" hypothesis for the electron transfer by Adx. The 1.5 A resolution crystal structure of a 1:1 Ru(bpy)2(mbpy)-Adx(1-108) complex reveals the site of modification, Cys95, and allows to predict the possible intramolecular electron-transfer pathways within the complex. Photoreduction of uncoupled Adx, mutant Adx(1-108), and Ru(bpy)2(mbpy)-Adx(1-108) using safranin T as the mediating electron donor suggests that two electrons are transferred from the dye to Adx. The intramolecular photoreduction rate constant for the ruthenated Adx has been determined and is discussed according to the predicted pathways.     

Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation

The 58-kDa complex formed between the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), and cytochrome P450cam (CYP101) from the bacterium Pseudomonas putida has been investigated by high-resolution solution NMR spectroscopy. Pdx serves as both the physiological reductant and effector for CYP101 in the enzymatic reaction involving conversion of substrate camphor to 5-exo-hydroxycamphor. In order to obtain an experimental structure for the oxidized Pdx-CYP101 complex, a combined approach using orientational data on the two proteins derived from residual dipolar couplings and distance restraints from site-specific spin labeling of Pdx has been applied. Spectral changes for residues in and near the paramagnetic metal cluster region of Pdx in complex with CYP101 have also been mapped for the first time using ¹⁵N and ¹³C NMR spectroscopy, leading to direct identification of the residues strongly affected by CYP101 binding. The new NMR structure of the Pdx-CYP101 complex agrees well with results from previous mutagenesis and biophysical studies involving residues at the binding interface such as formation of a salt bridge between Asp38 of Pdx and Arg112 of CYP101, while at the same time identifying key features different from those of earlier modeling studies. Analysis of the binding interface of the complex reveals that the side chain of Trp106, the C-terminal residue of Pdx and critical for binding to CYP101, is located across from the heme-binding loop of CYP101 and forms non-polar contacts with several residues in the vicinity of the heme group on CYP101, pointing to a potentially important role in complex formation.     

1H NMR identification of a beta-sheet structure and description of folding topology in putidaredoxin

Putidaredoxin (Pdx), a 106-residue globular protein consisting of a single polypeptide chain and a [2Fe-2S] cluster, is the physiological reductant of P-450cam, which in turn catalyzes the monohydroxylation of camphor by molecular oxygen. No crystal structure has been obtained for Pdx or for any closely homologous protein. The application of two-dimensional 1H NMR methods to the problem of structure determination in Pdx is reported. A beta-sheet consisting of five short strands and one beta-turn has been identified from distinctive nuclear Overhauser effect patterns. All of the backbone resonances and a majority of the side-chain resonances corresponding to protons in the beta-sheet have been assigned sequence specifically. The sheet contains one parallel and three antiparallel strand orientations. Hydrophobic side chains in the beta-sheet face primarily toward the protein interior, except for a group of three valine side chains that are apparently solvent exposed. The potential significance of this "hydrophobic patch" in terms of biological activity is discussed. The folding topology, as determined by the constraints of the beta-sheet, is compared with that of other [2Fe-2S] proteins for which folding topologies are known.     

Vertebrate-type and plant-type ferredoxins: crystal structure comparison and electron transfer pathway modelling

Crystallographic analysis of a fully functional, truncated bovine adrenodoxin, Adx(4-108), has revealed the structure of a vertebrate-type [2Fe-2S] ferredoxin at high resolution. Adrenodoxin is involved in steroid hormone biosythesis in adrenal gland mitochondria by transferring electrons from adrenodoxin reductase to different cytochromes P450. Plant-type [2Fe-2S] ferredoxins interact with photosystem I and a diverse set of reductases.A systematic structural comparison of Adx(4-108) with plant-type ferredoxins which share about 20 % sequence identity yields these results. (1) The ferredoxins of both types are partitioned into a large, strictly conserved core domain bearing the [2Fe-2S] cluster and a smaller interaction domain which is structurally different for both subfamilies. (2) In both types, residues involved in interactions with reductase are located at similar positions on the molecular surface and coupled to the [2Fe-2S] cluster via structurally equivalent hydrogen bonds. (3) The accessibility of the [2Fe-2S] cluster differs between Adx(4-108) and the plant-type ferredoxins where a solvent funnel leads from the surface to the cluster. (4) All ferredoxins are negative monopoles with a clear charge separation into two compartments, and all resulting dipoles but one point into a narrow cone located in between the interaction domain and the [2Fe-2S] cluster, possibly controlling predocking movements during interactions with redox partners. (5) Model calculations suggest that FE1 is the origin of electron transfer pathways to the surface in all analyzed [2Fe-2S] ferredoxins and that additional transfer probability for electrons tunneling from the more buried FE2 to the cysteine residue in position 92 of Adx is present in some.     

Infrared spectroscopic and mutational studies on putidaredoxin-induced conformational changes in ferrous CO-P450cam

Ferrous-carbon monoxide bound form of cytochrome P450cam (CO-P450cam) has two infrared (IR) CO stretching bands at 1940 and 1932 cm(-1). The former band is dominant (>95% in area) for CO-P450cam free of putidaredoxin (Pdx), while the latter band is dominant (>95% in area) in the complex of CO-P450cam with reduced Pdx. The binding of Pdx to CO-P450cam thus evokes a conformational change in the heme active site. To study the mechanism involved in the conformational change, surface amino acid residues Arg79, Arg109, and Arg112 in P450cam were replaced with Lys, Gln, and Met. IR spectroscopic and kinetic analyses of the mutants revealed that an enzyme that has a larger 1932 cm(-1) band area upon Pdx-binding has a larger catalytic activity. Examination of the crystal structures of R109K and R112K suggested that the interaction between the guanidium group of Arg112 and Pdx is important for the conformational change. The mutations did not change a coupling ratio between the hydroxylation product and oxygen consumed. We interpret these findings to mean that the interaction of P450cam with Pdx through Arg112 enhances electron donation from the proximal ligand (Cys357) to the O-O bond of iron-bound O(2) and, possibly, promotes electron transfer from reduced Pdx to oxyP450cam, thereby facilitating the O-O bond splitting.     

3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys204, Cys210, and Cys214 were individually replaced by Ser and in which Val207 was replaced by Cys were constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters were investigated. The FrdB Cys204Ser, Cys210Ser, and Cys214Ser mutations result in enzymes with negligible activity that have dissociated from the membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters, centers 3 and 2, respectively. In contrast, the FrdB Val207Cys mutation results in a functional membrane-bound enzyme that is able to support growth under anaerobic and aerobic conditions. However, EPR studies indicate that the indigenous [3Fe-4S]+,0 cluster (Em = -70 mV), center 3, has been replaced by a much lower potential [4Fe-4S]2+,+ cluster (Em = -350 mV), indicating that the primary sequence of the polypeptide determines the type of clusters assembled. The results of these studies afford new insights into the role of centers 2 and 3 in mediating electron transfer from menaquinol, the residues that ligate these clusters, and the intercluster magnetic interactions in the wild-type enzyme.     

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu, C.-A., Yu, L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Qo) site of the bc1 complex.