Barbara K. Burgess

An All-ferrous State of the Fe Protein of Nitrogenase INTERACTION WITH NUCLEOTIDES AND ELECTRON TRANSFER TO THE MoFe PROTEIN

The MoFe protein of nitrogenase catalyzes the six-electron reduction of dinitrogen to ammonia. It has long been believed that this protein receives the multiple electrons it requires one at a time, from the [4Fe-4S]2+/+ couple of the Fe protein. Recently an all-ferrous [4Fe-4S]0 state of the Fe protein was demonstrated suggesting instead a series of two electron steps involving the [4Fe-4S]2+/0 couple. We have examined the interactions of the [4Fe-4S]0 Fe protein with nucleotides and its ability to transfer electrons to the MoFe protein. The [4Fe-4S]0 Fe protein binds both MgATP and MgADP and undergoes the MgATP induced conformational change and then binds properly to the MoFe protein, as evidenced by the fact that the behavior of the 0 and +1 oxidation states in the chelation and chelation protection assays are indistinguishable. Nucleotide binding does not effect the distinctive UV/Vis, CD, or Mössbauer spectra exhibited by the [4Fe-4S]0 Fe protein; however, because the intensity of the g = 16.4 EPR signal of the [4Fe-4S]0 Fe protein is extremely sensitive to minor variations of the rhombicity parameter E/D, the EPR signal is sensitive to the binding of nucleotides. A 50:50 mixture of [4Fe-4S]2+ and [4Fe-4S]0 Fe protein results in electron self-exchange and 100% production of [4Fe-4S]+ Fe protein, demonstrating that the +1/0 couple is fully reversible. MgATP is absolutely required for electron transfer from the [4Fe-4S]0 Fe protein to the reduced state of the MoFe protein. In that reaction both electrons are transferred and are used to reduce substrate.

The FeMoco-deficient MoFe Protein Produced by a nifH Deletion Strain of Azotobacter vinelandii Shows Unusual P-cluster Features

The His-tag MoFe protein expressed by thenifH deletion strain Azotobacter vinelandiiDJ1165 (ΔnifH MoFe protein) was purified in large quantity. The α2β2 tetrameric ΔnifH MoFe protein is FeMoco-deficient based on metal analysis and the absence of the S = 3/2 EPR signal, which arises from the FeMo cofactor center in wild-type MoFe protein. The ΔnifH MoFe protein contains 18.6 mol Fe/mol and, upon reduction with dithionite, exhibits an unusually strong S = 1/2 EPR signal in the g ≈ 2 region. The indigo disulfonate-oxidized ΔnifH MoFe protein does not show features of the P2+ state of the P-cluster of the ΔnifB MoFe protein. The oxidized ΔnifH MoFe protein is able to form a specific complex with the Fe protein containing the [4Fe-4S]1+ cluster and facilitates the hydrolysis of MgATP within this complex. However, it is not able to accept electrons from the [4Fe-4S]1+ cluster of the Fe protein. Furthermore, the dithionite-reduced ΔnifH MoFe can be further reduced by Ti(III) citrate, which is quite unexpected. These unusual catalytic and spectroscopic properties might indicate the presence of a P-cluster precursor or a P-cluster trapped in an unusual conformation or oxidation state.

The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase

It is known that an E146D site-directed variant of the Azotobacter vinelandii iron protein (Fe protein) is specifically defective in its ability to participate in iron-molybdenum cofactor (FeMoco) insertion. Molybdenum-iron protein (MoFe protein) from the strain expressing the E146D Fe protein is partially (≈45%) FeMoco deficient. The “free” FeMoco that is not inserted accumulates in the cell. We were able to insert this “free” FeMoco into the partially pure FeMoco-deficient MoFe protein. This insertion reaction required crude extract of the ΔnifHDK A. vinelandii strain CA12, Fe protein and MgATP. We used this as an assay to purify a required “insertion” protein. The purified protein was identified as GroEL, based on the molecular mass of its subunit (58.8 kDa), crossreaction with commercially available antibodies raised against E. coli GroEL, and its NH2-terminal polypeptide sequence. The NH2-terminal polypeptide sequence showed identity of up to 84% to GroEL from various organisms. Purified GroEL of A. vinelandii alone or in combination with MgATP and Fe protein did not support the FeMoco insertion into pure FeMoco-deficient MoFe protein, suggesting that there are still other proteins and/or factors missing. By using GroEL-containing extracts from a ΔnifHDK strain of A. vinelandii CA12 along with FeMoco, Fe protein, and MgATP, we were able to supply all required proteins and/or factors and obtained a fully active reconstituted E146D nifH MoFe protein. The involvement of the molecular chaperone GroEL in the insertion of a metal cluster into an apoprotein may have broad implications for the maturation of other metalloenzymes.

Discovery of a Novel Ferredoxin from Azotobacter vinelandii Containing Two [4Fe-4S] Clusters with Widely Differing and Very Negative Reduction Potentials

Ferredoxins that contain 2[4Fe-4S]2+/+ clusters can be divided into two classes. The “clostridial-type” ferredoxins have two Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro motifs. The “chromatium-type” ferredoxins have one motif of that type and one more unusual Cys-Xaa-Xaa-Cys-Xaa7–9-Cys-Xaa-Xaa-Xaa-Cys-Pro motif.Here we report the purification of a novel ferredoxin (FdIII) from Azotobacter vinelandii which brings to 12 the number of small [Fe-S] proteins that have now been reported from this organism. NH2-terminal sequencing of the first 56 amino acid residues shows that FdIII is a chromatium-type ferredoxin with 77% identity and 88% similarity to Chromatium vinosumferredoxin. Studies of the purified protein by matrix-assisted laser desorption ionization–time of flight mass spectroscopy, iron analysis, absorption, circular dichroism, and electron paramagnetic resonance spectroscopies show that FdIII contains 2[4Fe-4S]2+/+clusters in a 9,220-Da polypeptide. All 2[4Fe-4S]2+/+ferredoxins that have been studied to date, including C. vinosum ferredoxin, are reported to have extremely similar or identical reduction potentials for the two clusters. In contrast, electrochemical characterization of FdIII clearly establishes that the two [4Fe-4S]2+/+ clusters have very different and highly negative reduction potentials of −486 mV and −644 mVversus the standard hydrogen electrode.

Structure and Reactivity of Nitrogenase — An Overview

Nitrogenase is composed of two separately purified proteins called the molybdenum-iron protein (MoFe protein) and the iron protein (Fe protein).1. N2 fixation and all other reductions catalyzed by nitrogenase require both component proteins, a source of reducing equivalents, MgATP, protons and an anaerobic environment. This overview covers what has been learned recently about the composition, structure and redox properties of the two component proteins and the events that occur during nitrogenase turnover. Fortunately, diffraction quality crystals have recently been obtained for both the MoFe protein (Cp1 and Av1; Weiniger, Mortenson, 1982) and the Fe protein (Av2; Rees, Howard, 1983). Thus, many of the questions raised below may soon have definitive answers.

Azotobacter vinelandii NADPH:Ferredoxin Reductase Cloning, Sequencing, and Overexpression

Azotobacter vinelandii ferredoxin I (AvFdI) controls the expression of another protein that was originally designated Protein X. Recently we reported that Protein X is a NADPH-specific flavoprotein that binds specifically to FdI (Isas, J. M., and Burgess, B. K.(1994) J. Biol. Chem. 269, 19404-19409). The gene encoding this protein has now been cloned and sequenced. Protein X is 33% identical and has an overall 53% similarity with the fpr gene product from Escherichia coli that encodes NADPH:ferredoxin reductase. On the basis of this similarity and the similarity of the physical properties of the two proteins, we now designate Protein X as A. vinelandii NADPH:ferredoxin reductase and its gene as the fpr gene. The protein has been overexpressed in its native background in A. vinelandii by using the broad host range multicopy plasmid, pKT230. In addition to being regulated by FdI, the fpr gene product is overexpressed when A. vinelandii is grown under N2-fixing conditions even though the fpr gene is not preceded by a nif specific promoter. By analogy to what is known about fpr expression in E. coli, we propose that FdI may exert its regulatory effect on fpr by interacting with the SoxRS regulon.

The Role of Methionine 156 in Cross-subunit Nucleotide Interactions in the Iron Protein of Nitrogenase

A variant Fe protein has been created at the completely conserved residue methionine 156 by changing it to cysteine. The Azotobacter vinelandii strain expressing M156C is unable to grow under nitrogen-fixing conditions, and the purified protein cannot support substrate reduction in vitro. This mutation has an effect on the Fe protein’s ability to undergo the MgATP-induced conformational change as evidenced by the fact that M156C is chelated in the presence of MgATP with a lower observed rate than wild-type. While the electron paramagnetic resonance spectra of this protein are similar to those of the wild-type Fe protein, the circular dichroism spectrum is markedly different in the presence of MgATP, showing that the conformation adopted by M156C following nucleotide binding is different from the wild-type conformation. Although competition activity and chelation assays show that this Fe protein can still form a complex with the MoFe protein, this altered conformation only supports MgATP hydrolysis at 1% the rate of wild-type Fe protein. A model based on x-ray crystallographic information is presented to explain the importance of Met-156 in stabilization of the correct conformation of the Fe protein via critical interactions of the residue with Asp-43 and nucleotide in the other subunit.

Complex formation between Azotobacter vinelandii ferredoxin I and its physiological electron donor NADPH-ferredoxin reductase.

In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPH-ferredoxin reductase (FPR). In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined. AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI. The Lys in FPR has been identified as Lys258, a residue that forms a salt bridge with one of the phosphate oxygens of FAD in the absence of FdI. UV-Vis and circular dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region close to the FAD. Cytochrome c reductase assays and electron paramagnetic resonance data show that electron transfer between the two proteins is pH-dependent and that the [3Fe-4S]+ cluster of FdI is specifically reduced by NADPH via FPR, suggesting that the [3Fe-4S] cluster is near FAD in the complex. To further investigate the FPR:FdI interaction, the electrostatic potentials for each protein were calculated. Strongly negative regions around the [3Fe-4S] cluster of FdI are electrostatically complementary with a strongly positive region overlaying the FAD of FPR, centered on Lys258. These proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are physiological redox partners.

Alteration of the Reduction Potential of the [4Fe-4S]2+/+ Cluster of Azotobacter vinelandii Ferredoxin I

The [4Fe-4S]2+/+ cluster ofAzotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E 0′) relative to other structurally similar ferredoxins. Previous attempts to raise thatE 0′ by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter theE 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, theE 0′ increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control ofE 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 Å, 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 inE 0′.

Reconstitution of Azotobacter vinelandii ferredoxin I as a {2[4Fe-4S]1+/2+} protein

As normally isolated, Azotobacter vinelandii ferredoxin I (Fd I) is a {[4Fe‐4S],[3Fe‐3S]} protein: (7Fe)Fd I. We report that anaerobic reconstitution of Fd I from its apoprotein yields a protein whose spectra are distinct from those of (7Fe)Fd I and typical of bacterial ferredoxins. We identify this new form of Fd I as a {2[Fe‐4S]} protein: (8Fe)Fd I. (8Fe)Fd I is unstable in air and decomposes to give a ∼ 10% yield of (7Fe)Fd I. These results increase the probability that (8Fe)Fd I is the form of Fd I occurring in vivo and that (7Fe)Fd I results from oxidative degradation during purification.