Azotobacter vinelandii nitrogenase: “Kinetics of nif gene expression and insights into the roles of FdxN and NifQ in FeMo-co biosynthesis”

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(1)! ! UNIVERSIDAD POLITÉCNICA DE MADRID ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS DEPARTAMENTO DE BIOTECNOLOGÍA. !. TESIS DOCTORAL. Azotobacter vinelandii nitrogenase: “Kinetics of nif gene expression and insights into the roles of FdxN and NifQ in FeMo-co biosynthesis”. Autor: Emilio Jiménez Vicente Director: Dr Luis Rubio Herrero.

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(3) ! ! UNIVERSIDAD POLITÉCNICA DE MADRID ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS DEPARTAMENTO DE BIOTECNOLOGÍA. Memoria Presentada por D. Emilio Jiménez Vicente para optar al grado de Doctor. Director Dr. Luis Rubio Herrero Profesor Titular UPM. Madrid, 2014. ! This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognize that its copyright rest with the author and that no quotation from the thesis, or any information derived therefrom may be published without the author’s prior, written consent..

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(8) ! !. Abstract ! The Molybdenum-nitrogenase is responsible for most biological nitrogen fixation activity (BNF) in the biosphere. Due to its great agronomical importance, it has been the subject of profound genetic and biochemical studies. The Mo nitrogenase carries at its active site a unique iron-molybdenum cofactor (FeMoco) that consists of an inorganic 7 Fe, 1 Mo, 1 C, 9 S core coordinated to the organic acid homocitrate. Biosynthesis of FeMo-co occurs outside nitrogenase through a complex and highly regulated pathway involving proteins acting as molecular scaffolds, metallocluster carriers or enzymes that provide substrates in appropriate chemical forms. Specific expression regulatory factors tightly control the accumulation levels of all these other components. Insertion of FeMo-co into a P-cluster containing apo-NifDK polypeptide results in nitrogenase. reconstitution.. Investigation. of. FeMo-co. biosynthesis. has. uncovered new radical chemistry reactions and new roles for Fe-S clusters in biology.. !. %!.

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(13) ! !. Abbreviations ! 2-OG. 2-oxoglutarate. ADP. adenosine diphosphate. ATP. Adenosine triphosphate. Blast. Basic Local Alignment Search Tool. BNF. Biological nitrogen fixation. CFU. Colony-Forming Units. cGMP DNA. Desoxiribonucleic acid. DTH. Sodium dithionite. EBP. Enhancer Binding Protein. EPR. Electronic Paramagnetic Resonance. ESE. Electro Epin Echo. EXAFS FAD. Extended X-ray Absorption Fine Structure Flavin-adenine-dinucleotide. FeFe-co. Iron-iron cofactor. FeMo-co. Iron-molybdenum cofactor. FeV-co GAF GOGAT. Iron-anadium cofactor cGMPphosphodiesterase adenylate cyclase FhlA Glutamine oxoglutarate aminotransferase. HPK. Histidine Protein Kinase. IHF. Integration Host Factor. Mo-co. Molybdenum cofactor. MoSto. Dinitrogenase Molybdenum Storage protein. mRNA. Messenger Ribonucleic acid. NAS. Normalized Absolute Signal. MoFe protein. nif NifB-co. gene designation for molybdenum-dependent nitrogen fixation NifB cofactor. NMF. N-Methyl-Formamide. NMR. Nuclear Magnetic Resonance. NRVS. Nuclear Resonance Vibrational Spectroscopy. OD600. Optical Density at 600nm. ORF. Open Reading Frame. PAS. PLP. Per (period circadian protein) Arnt (aryl hydrocarbon receptor nuclear translocator protein) Sim (singleminded protein) Pyridoxal phosphate. qRT. Quantitative Reverse Transcriptase. RNA. Ribonucleic acid. rnf. !. Ciclic guanosine monophosphate. Genes homologous to Rhodobacter capsulatus nif genes. .!.

(14) ! ! RPM. rounds per minute. SAM. S-Adenosyl Methionine. UAS. Upstream Activation Sequence. UMP. Uridine MonoPhosphate. UR. uridylyl-removing. UTase. uridylyltransferase. UV XAS. Ultraviolet X-ray Absorption Spectroscopy. !. !. "/!.

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(16) ! !. Chapter 1: Introduction ! ! ! ! ! ! ! ! !. "#!.

(17) ! ! ! !. "#"!$%&'()*+,-!,.!-+*/,&%-!.+0)*+,-!1"#$2!&%-%3! ! %&%&%!4%-%*+5!,/&)-+6)*+,-!,.!"#$!&%-%3!+-!'&(#")*+",##! ! The nif regulon is defined as nifA and those genes under the control of NifA that are responsible for the production of a functional Mo nitrogenase. In Azotobacter vinelandii, nif genes are clustered in two chromosomal regions adjacent and equidistant from the replication origin [1] comprising at least eight operons [2-5] (Fig.1). From a genetic standpoint, such a position suggests a higher gene dosage during active cell growth [1] or just a critical role of these genes in A. vinelandii life style. Interestingly, all genes known to be required for the Mo, V and Fe-only nitrogenases (e.g. nifU, nifS, nifV, nifM, and nifB) are located in Mo nitrogenase regions [4-7]. The so-called major nif cluster contains at least six transcriptional units, namely orf12orf13, nifHDKTY, nifENX, orf5, iscAnifnifUSVcysE1nifnifWZMclpX2 and nifF, in which nif genes are interspersed with a number of open reading frames (ORFs) of unknown function [4]. The minor nif cluster contains three operons, namely rnfABCDGEH, nifLA and nifB fdxN nifOQ rhdN grx5nif, and the nafY gene [5, 8-10]. Genes in nif operons are transcribed by #54-containing RNA polymerase, that recognizes promoter DNA with boxes centered at around -12 and -24 from the transcription start point with a consensus sequence of 5'-YTGGCACGR-N3TTGCW-3' [11, 12]. Transcription by #54-containing polymerases is dependent of additional transcriptional factors denominated Enhancer Binding Proteins (EBPs), bind to DNA regions known as Upstream Activator Sequences (UAS) to initiate RNA synthesis [13]. The two EBPs NtrC and NifA are responsible for regulating the transcription of genes involved in the assimilation of N2. NtrC is well characterized in diazotrophic enteric bacteria as involved in nif genes transcription, meanwhile, nif gene expression in A.vinelandii is NifA-dependent !. "$!.

(18) ! ! but NtrC-independent, where NtrC has been shown to be responsible of activation of genes involved in assimilation of other N2 sources such as nitrate [14]. There are cases in which additional DNA binding proteins are needed, including the integration host factor [15]. IHF is an asymmetric histone-like protein that binds and bends DNA in specific locations (IHF binding consensus sequence 5'-WATCAANNNNTTR-3') [16]. Despite of the fact that only 11 gene products form the core of Mo nitrogenase dependent N2 fixation system (nifH, nifD, nifK, nifE, nifN, nifB, nifU, nifS, nifV, nifQ, and nifM) a genome-wide transcription profile analysis (comparing steady-state gene expression levels in N2-fixing A. vinelandii cells versus NH3 assimilating cells) demonstrated that ca. 400 genes were differentially expressed under Mo-dependent diazotrophic growth conditions [17, 18]. Recently, a synthetic biology approach to engineer nitrogenase in Klebsiella pneumonae was constructed by using 20 genes [19]. The low level of nitrogenase activity obtained suggest that synthetic N2 fixation operons need not only to balance gene expression but also to adjust it overtime. Uncoordinated expression or inadequate Nif protein concentration might be deleterious to N2 fixation.. & & & !. "%!.

(19) ! ! & & &. !" A. vinelandii chromosome %!#$. &!#$ !"#$. #" major nif cluster. H. D. K. T Y. E. N. iscAnif U. X. S. V cysE W Z M clpX2. minor nif cluster nifOQ grx5nif nafY H E G. D. C. B A. L. A. B fdxN. rhdN. rnf. & & (478/9&B2&'/70>4Z0.45>&56&>46&79>9&4>&"2&=4>9;0>E44&I"J&$510.45>&56&!"#W&.!#&0>E& )!#-5A9/5>-& 4>& $1-."!+/)!0""& 1@/5M5-5M92 (B) A. vinelandii Mo-nitrogenase nif gene clusters. Predicted "54–dependent promoter regions are depicted by !. "&!. F.

(20) ! ! arrows. Black arrows represent regions additionally containing NifA-UAS and IHF motifs; blue arrows represent regions containing NifA-UAS motifs but lacking IHF motifs; orange arrows represent regions lacking both NifA-UAS and IHF motifs. Adapted from [20].. "#"#7!89%!:+.;<:+.=<4(->!3?3*%@A!)!B?-)@+5!3+&-)(!+-*%&/)*+-&!3?3*%@!*9)*! @,B'()*%3!"#$!&%-%!%0C/%33+,-! ! N2 fixation is a very high energy demanding process. Thus, a strictly regulated mechanism that control nitrogenase synthesis and activity is mandatory in order to minimize waste of energy. The NifA-NifL-GlnK complex is key in regulating nif gene expression. In A. vinelandii this system integrates different environmental and intracellular signals including cell energy levels (ATP/ADP ratio), N2/carbon balance, and redox state, to determine if diazotrophic growth is feasible. Different diazotrophic proteobacteria, such as A. vinelandii, K. pneumoniae and Pseudomonas stutzeri, display significant differences on their signal integration mechanisms [21-24]. The biochemical basis of the NifA-NifL-GlnK complex relies on a series of sensing and interaction motifs that have been studied in detail (Fig. 2A) [2527]. NifL is an evolutionary relative of Histidine Protein Kinases (HPK) that acts as anti-activator of NifA. NifL domain architecture is similar to cytoplasmic HPKs. It contains two N-terminal PAS motifs (Per-ARNT-Sim) [28, 29] and a Cterminal transmitter region containing a conserved H domain that acts as signal integrator and a nucleotide binding domain belonging to GHKL (Gly-His-LysLeu) superfamily of ATPases [30, 31]. PAS1 carries a flavin-adeninedinucleotide (FAD) cofactor. FAD cofactor oxidation results in activation of NifL and consequent inhibition of NifA activity, whereas reduction of the FAD moiety deactivates NifL [28]. The H domain of NifL is located between the PAS2 and GHKL domains. Although the conserved His is not essential, other amino acids !. "'!.

(21) ! ! from the H domain play an important role in signal transduction associated with conformational changes that modulate the interaction of NifL with the 2oxoglutarate(2-OG)-bound form of NifA [32-34]. The NifL GHKL ATP-binding domain, does not hydrolyze ATP, however it seems to perform two different functions: (i) it senses the energy status of the cell by binding ADP, and (ii) it senses the cellular N2 level through an interaction with GlnK, a PII-like protein [25, 35, 36]. As mentioned before, NifA is an EBP that activates transcription of "54dependent promoters [12]. The N-terminal part of NifA presents a GAF domain (cGMPphosphodiesterase adenylate cyclase FhlA domain) that binds 2-OG for allosteric control of NifA. Interaction of 2-OG with the GAF domain of NifA prevents NifL interaction in the absence of unmodified GlnK [34, 37]. 2-OG is a Krebs cycle intermediate that provides, not just a direct measure of cellular carbon status, but also an indirect measure of cellular N2 status. This is because 2-OG is a substrate of the Glutamine oxoglutarate aminotransferase (GOGAT) enzyme and a main source of carbon skeletons for amino acid biosynthesis [38]. Additionally, NifA presents a central catalytic AAA+ domain that can interact with the "54 RNA polymerase subunit [39]. Binding of NifL inhibits the ATPase activity of this domain disabling nif gene expression [34, 40]. The C-terminal part of NifA includes a helix-turn-helix DNA-binding domain that recognizes NifA-UAS located 5' of nif genes. When A. vinelandii cells are growing under ideal diazotrophic conditions (N2 starvation and high respiratory rates) the level of oxygen is low and the levels of 2-OG and ATP are high. Allosteric binding of 2-OG to the GAF domain produces a conformational change in NifA that impairs NifL binding. Free NifA is able to activate nif gene expression through DNA binding and activation of the "54 factor. On the other hand, at relatively low 2-OG levels or, most importantly, when NifL FAD group is oxidized by excess oxygen, NifL is competent enough to bind NifA and inhibit its activity. However, additional fine-tuning applies that will be described below [22].. !. "(!.

(22) ! ! A. vinelandii relies on GlnK and GlnD to determine its cellular carbon/N2 balance [41]. The glnK gene product is a member of the PII family that stabilizes NifL-inhibition of NifA by forming a GlnK-NifL-NifA ternary complex in response to NH4+ excess. Contrary to the well-studied case of enteric bacteria, including the diazotrophic enterobacterium K. pneumoniae, GlnK expression in A.vinelandii appears to be independent of NtrC [41]. GlnK is susceptible to be urydilated or de-urydilated by GlnD (an uridylyltransferase/uridylyl-removing enzyme). The activity of GlnD depends of glutamine concentration, at high concentration, binding of glutamine to the enzyme switches activity in favor of uridyl-removing activity, whereas low glutamine concentration switches activity toward an uridylyltransferase function [42, 43]. Uridylylation of GlnK by GlnD plays a key role in nif gene activation of expression [25]. Under conditions of N2 excess (high N2/carbon ratio) resulting in high concentration of glutamine, non-uridylylated GlnK blocks the ammonium transporter AmtB [44]. It also interacts with the C-terminal domain of NifL increasing its inhibitory effect over NifA and forming a ternary NifL-NifA-GnlK complex that is more stable to dissociation by binding of 2-OG than the NifLNifA complex (Fig. 2B). Thus, the N2 signal overrides the carbon source signal. On the other hand, under conditions of N2 limitation (low N2/carbon balance) GlnD uridylylates GlnK (GlnK-UMP3), which cannot interact with NifL. In addition, 2-OG binding to NifA prevents its inhibition by NifL (Fig. 2B). Interestingly, there is no evidence of NifL interaction with cytoplasmic membranes in A. vinelandii as it has been shown to be the case in K. pneumonia, where NifL could be immobilized in the membrane, and therefore, hijacked from NifA, resulting in activation of nif transcription [45].. !. ")!.

(23) ! !. #!. GlnK. P P FAD/H2 PAS1. H. GHKL. Signal integrator. Nitrogen and Energy status. PAS2. redox sensor. NifL. ADP. 2-OG. GAF. AAA+. HTH. N/C status. Interacts with !54" ATPase activity. DNA-binding domain". Nitrogen excess. %!. NifA. Nitrogen starvation NH4+. Amtb. Amtb GlnK. [2-OG]. [Gln]. FADH2. GlnD. NifL. UR UTase. GlnK GlnK-UMP3. P P FAD. ADP. NifL. 2-OG. NifA. NifA !"#$!. !54. nif. !$$#. ".!. 2NH3+H2. Nitrogenase nif. !"#. &. !. "%&###"'&#(#&)#. N2+8e-+8H+.

(24) ! ! && &(478/9& F2& !@9& *46"[*46$[P;>\& /978;0.5/L& -L-.9M& 56& $1- ."!+/)!0""1 (A) Schematic domain architecture of A. vinelandii NifL and NifA proteins. (B) GlnKNifL-NifA response to environmental and metabolic conditions in A. vinelandii. Left panel: conditions of N2 excess result in high concentration of glutamine that leads to deuridylylation of GlnK by the uridylyl-removing (UR) activity of GlnD. The unmodified form of GlnK can interact with (1) AmtB to block active transport of NH3, and (2) with NifL in a GlnK-NifL-NifA ternary complex to block activation of nif gene transcription. Right panel: conditions of N2 limitation result in low glutamine and high 2-OG concentrations that lead to uridylylation of GlnK (GlnK-UMP3) by the uridylyltransferase (UTase) activity of GlnD. The modified form of GlnK is unable to interact with either AmtB or NifL. In addition, high 2OG levels induce a conformational change in NifA that prevents NifL inhibition and allows binding to specific UAS activating nif gene transcription. Adapted from [20].. ! ! ! ! ! ! !. !. #/!.

(25) ! ! !. "#7!D+,3?-*9%3+3!,.!@%*)(!5('3*%/!,.!*9%!@,(?EB%-'@!-+*/,&%-)3%!! !. "#7#"!4%-%/)(!5,-5%C*3! ! Two different major strategies for metal cofactor biosynthesis can be found in nature. In some cases, the cofactor is assembled directly in the final target protein. The nitrogenase [8Fe-7S] P-cluster is an example of proteins where in situ cofactor assembly takes place [46-50]. In the case of more complex prosthetic groups, the opposite approach is used. The ironmolybdenum cofactor (FeMo-co) of nitrogenase, the molybdenum cofactor (Moco) of nitrate reductase, and the H-cluster of [FeFe]-hydrogenase are examples of cofactors where ex situ assembly occurs [51-53].. FeMo-co synthesis is. completed outside the target enzyme in a biosynthetic pathway completely independent of the production of the structural polypeptides. Thus, FeMo-co needs to be inserted into an apo-enzyme in order to render the mature, active nitrogenase enzyme. !. "#7#7!F+-+*/,&%-)3%!)-B!)C,<B+-+*/,&%-)3%! ! NifDK (also referred to as dinitrogenase or MoFe protein or nitrogenase component I) is a 230-kDa $2%2 tetramer of the nifD and nifK gene products. The $ and % subunits arrange as a pair of $% dimers that are related by a twofold rotation axis. Both $ and % subunits (NifD and NifK respectively) are phylogenetically related and display a similar tertiary structure consisting of three domains each. NifDK contains two unique metal clusters per $%-dimer: the P-cluster and the FeMo-co [54].. !. #"!.

(26) ! ! The P-cluster is a [8Fe-7S] cluster in which two [4Fe-4S] cubanes share a µ6-S atom. The P-clusters are located at the interface between the $ and % subunits at around 12 Å below the protein surface. In the DTH-reduced state of NifDK, residues $-Cys88 and %-Cys95 provide thiol groups bridging the two cubanes, whereas residues $-Cys62, $-Cys154, %-Cys70 and %-Cys153 coordinate the remaining Fe sites in the P-cluster (residue numbers correspond to the A. vinelandii NifDK). NifU and NifS are needed for the initial formation of two pairs of [4Fe-4S] clusters that serve as precursors to the P-clusters. The concerted action of both NifZ and NifH is required for the biosynthesis of the complete set of P-clusters, which will be carried out in situ (directly on the NifDK protein). A NifDK protein carrying only one P-cluster and one pair of [4Fe-4S] cluster or with two pairs of [4Fe-4S] clusters (but no P-clusters) is obtained from deletion mutants lacking NifZ or NifH, respectively [55]. The FeMo-co comprises a [Mo-7Fe-9S] cluster with a single carbide atom residing in the cavity formed by the six central Fe atoms [56-58]. In addition, the Mo atom is coordinated by the C-2 carbonyl and hydroxyl groups of the organic acid homocitrate (Fig. 3). FeMo-co is almost completely buried within the $-subunit of NifDK, 10 Å below the protein surface and 14 Å away from the P-cluster. Hydrophilic residues form the majority of the protein environment around FeMo-co, although a number of hydrophobic residues are required for cofactor positioning. Unlike the P-cluster, FeMo-co is ligated by only two NifD amino acid residues: $-His442 (which binds to the Mo atom) and $Cys275 (which binds to the Fe atom located at the opposite end of the cluster) [54]. Several other residues surrounding the cofactor binding site are selected to create a protein environment tailored for FeMo-co binding, such as $-Gly356 and $-Gly357 (which are needed to prevent steric hindrance with the metal cluster), $-Arg96 and $-Arg359 (which hydrogen bond to and stabilize the cofactor), or $-Gln191, $-Glu440 and $-Glu427 (which interact directly or through water molecules with the homocitrate moiety). As expected, residues $-His442,. !. ##!.

(27) ! ! $-Cys275 and some other residues in the vicinity of FeMo-co are highly conserved across species.. & & & !. #$!.

(28) ! ! & (478/9&K2&".5M41&-./81.8/9&56&(9R5Q152 Adapted from [51]. Apo-NifDK (also referred to as apo-dinitrogenase or apo-MoFe protein) is a cofactor-less NifDK protein. Several forms of apo-NifDK have been reported to accumulate in the cell depending on the genetic background [47]. For example, a "nifH mutation renders apo-NifDK lacking both the P-clusters and FeMo-co whereas a "nifB mutation renders apo-NifDK that contains P-clusters but lacks FeMo-co. Here we will use the term apo-NifDK when referring to the FeMo-co less form found in "nifB strains [56], otherwise a more detailed definition will be included. This form of apo-NifDK can be readily activated by the simple addition of FeMo-co. In fact, apo-NifDK activation was used as assay to isolate FeMo-co from pure preparations of NifDK protein [59, 60]. In general, minor differences are observed in apo-NifDK structure upon FeMo-co insertion. However, the $III domain undergoes major structural rearrangements. A comparison between the apo- and holo-NifDK structures revealed a His triad ($-His274, $-His442 and $-His451) possibly involved in the formation of an insertion funnel in the structure of "nifB NifDK. The rearrangement of the $III domain is hypothesized to generate an opening for FeMo-co insertion and to provide a positively charged path to drive FeMo-co entrance down to the cofactor binding site [56] (Fig. 4). Site-directed mutagenesis studies on NifDK are consistent with the important role of the histidine residues along the insertion funnel to facilitate FeMo-co insertion [55].. !. #%!.

(29) ! !. !". #". +(,-"#-*. !"#$%&'()*. (478/9& 32& +./81.8/9-& 56& *46]\& 0>E& 0A5Q*46]\2 (A) Structure of one NifDK #$ pair. (B) Structure of one apo-NifDK #$ pair. Color code: # subunits; #I domain in cyan, #II domain in purple and #III domain in orange. % subunits; $I domain in grey, $II domain in green and $III domain in wheat. The #III domain estructure shows diferent $ sheet orientation in NifDK and apo-NifDK forms. The cavity formed between domains #I, #II and #III is occupied by FeMo-co in NifDK, whereas this cavity is empty in apo-NifDK.. !. #&!.

(30) ! !. "#7#G!H%I,<5,!D+,3?-*9%3+3! ! The isolation of FeMo-co from pure preparations of NifDK protein is one of the seminal contributions to the field of nitrogenase biochemistry and to our understanding of complex metalloproteins assembly in general. FeMo-co isolation set the basis for the in vitro FeMo-co insertion and FeMo-co synthesis and insertion assays developed by Vinod Shah and now widely used in the field [60]. Purified FeMo-co was found to be extremely sensitive to oxygen and unstable in protic solvents. Thus, FeMo-co extraction must be carried out into anaerobic N-methyl formamide (NMF) after denaturing and precipitating pure NifDK in a series of low and neutral pH solutions. FeMo-co isolated in this manner is stable indefinitely when stored as an anaerobic NMF solution under liquid nitrogen. Combined genetic and biochemical studies have determine that FeMo-co biosynthesis requires (1) enzymes to provide substrates in the appropriate chemical forms and to catalyze certain critical reactions such as carbide insertion, (2) molecular scaffolds to aid in the step-wise assembly of FeMo-co, and (3) metallocluster carrier proteins that escort FeMo-co biosynthetic intermediates in their transit between scaffolds (Fig 5) (Table 1) [51]. Once fully assembled, FeMo-co is transferred from the “FeMo-co biosynthetic factory” into apo-NifDK, either by a hypothetical protein-protein interaction between NifEN and apo-NifDK [55] or mediated by the FeMo-co binding protein NafY [51]. The insertion of FeMo-co into apo-NifDK generates mature, functional holo-NifDK. The specific roles of number of Nif proteins in FeMo-co biosynthesis are described below:. !. #'!.

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email protected]! 3$!)-5@*$',-$!9*%B!D*2PF!2-&5(!'!()';-.<!+&-%#*$! *$/-./#<!*$!.'%#&!(%#+(!-2!>#L-1)-!'((#[email protected]! G-4@.#!&-.#!(%'@*.*R*$"!'+-1D*2GJ!'$<!*$!>#L-1)-! *$(#&,-$! 3$/-./#<!*$!S1).4(%#&!2-&5',-$!. G*$*%&-"#$'(#!KL->#!+&-%#*$M!T1(4@4$*%6!>#L-1)-!*(! @4&*#<!9*%B*$!%B*(!(4@4$*%! G*$*%&-"#$'(#!KL->#!+&-%#*$M!U1(4@4$*%!-26!S1).4(%#&!*(! !"#5% .-)'%#<!'%!%B#!*$%#&2')#!-2!T!'$<!U!(4@4$*%(! V#W4*&#<!2-&!5'%4&',-$!-2!D*2O!K>#!+&-%#*$M6!:*5*.'&!%-! !"#6% +&-.A.!*(-5#&'(#(! G*$*%&-"#$'(#!&#<4)%'(#!K>#!+&-%#*$M6!X@.*"'%#!#.#)%&-$! !"#7% <-$-&!%-!D*2GJ6!0.(-!&#W4*&#<!2-&!S1).4(%#&!'$<!>#L-1 )-!@*-(A$%B#(#(6! !"#4%. !0,;9B2&G5;9&56&>46&79>9&A/5E81.-&4>&R5Q>4./579>0-92 Genes from the N2-fixing bacterium A. vinelandii are listed. The color code is as follows: green, genes !. #(!.

(32) ! ! required in vitro for Mo-nitrogenase synthesis or activity; red, genes required in vivo for the Mo-nitrogenase and also for the alternative V and Fe-only nitrogenases. Adapted from [61].. "#7#J!:+.K!)-B!:+.L! ! Many of the proteins involved in N2 fixation, including nitrogenase itself, are iron-sulfur (Fe-S) proteins. Given the large amount of Nif proteins expressed in N2-fixing conditions, a specific [Fe-S] cluster biosynthetic system is found in diazotrophic microorganisms in addition to the general [Fe-S] cluster biosynthetic machinery. This redundancy presents at least two major advantages: (i) it satisfies the high demand of [Fe-S] clusters needed for N2 fixation and (ii) it ensures that a deleterious mutation disturbing this specialized system only affects cell survival under diazotrophic growth conditions [46]. NifU and NifS are required for the maturation of both NifH and NifDK. The critical observation to identify a role for these proteins was that whereas many mutations in nif genes affected either NifH or NifDK, mutations in either nifU or nifS resulted in a large decrease of activity in both nitrogenase components [4]. Since the presence of [Fe-S] clusters is common to both components it was proposed that NifU and NifS proteins had a role in the assembly of nitrogenase-specific [Fe-S] clusters. An additional key observation was the fact that, although nitrogenase activity was severely affected in nifU and nifS mutants, it was not completely lost. This led to the identification of additional housekeeping NifU and NifS homologues, referred to as IscU and IscS, which were involved in supplying [Fe-S] clusters for general cellular functions [62]. NifS is a 87-kDa pyridoxal phosphate-containing (PLP) homodimer [63]. NifS is a cysteine desulfurase that catalyzes a desulfurization reaction of Lcysteine, rendering L-alanine and a protein-bound persulfide as products. A highly conserved Cys325 residue located in the active site of the enzyme was found critical for NifS activity [64]. Based on amino acid sequences, cysteine !. #)!.

(33) ! ! desulfurases are classified in two major groups; NifS has a SSGSAC(T/S)S conserved consensus sequence and falls within group I [50]. Interestingly, a cysE homologue, encoding an O-acetyl serine synthase, the rate-limiting step for cysteine biosynthesis, is co-transcribed together with nifS [65]. NifU is a 66-kDa homodimer containing a stable [2Fe-2S] cluster per subunit [66]. Amino acid sequence conservation analysis, site-directed mutagenesis experiments and activity assays with separate purified domains confirmed the presence of three conserved domains in NifU [67]. The central domain contains a permanent, redox-active, [2Fe-2S] cluster coordinated by four conserved cysteine residues, whereas the N-terminal and C-terminal domains present three and two conserved cysteine residues, respectively, for the assembly of transient [Fe-S] clusters [68]. Spectroscopic and genetic analyses provided further evidence of formation of labile [2Fe-2S] clusters within both terminal domains of NifU in reactions containing L-cysteine, Fe2+ and NifS [69]. A series of elegant experiments using apo-NifH as [Fe-S] cluster acceptor provided further details on the mechanism of NifS/NifU [64, 70]. NifS activity directs the assembly of transient [4Fe-4S] clusters on NifU, which are subsequently transferred to apo-NifH endowing protein activity. A NifUS complex formed during cluster assembly has been reported [69], but NifS was not required for [Fe-S] cluster transfer from NifU to the target apo-protein. Although in vitro loading of apo-NifH with [4Fe-4S] clusters was possible simply by incubating apo-NifH with Fe2+ and S2-, the reaction was significantly faster (at physiologically significant rates), specific and more efficient (requiring only equimolar amounts of NifU) when using NifS, Fe2+ and L-cysteine. All together, these results confirmed the role of NifS as donor of S2- in order to sequentially load the scaffold NifU for the synthesis of simple [2Fe-2S] and [4Fe-4S] clusters required for maturation of nitrogenase components. NifU and NifS are also involved in FeMo-co synthesis (Fig. 5). Participation of these proteins as providers of [Fe-S] cluster substrates for FeMo-co biosynthesis was difficult to demonstrate because many FeMo-co !. #.!.

(34) ! ! biosynthetic proteins are Fe-S proteins themselves and mutations in nifU or nifS would have pleiotropic effects on the pathway. This puzzle was solved by investigating the capability of nifUS double mutants to synthesize NifB-co, an early precursor to FeMo-co (see below). NifB-co biosynthesis was practically abolished in nifUS mutants [71]. Because NifU and NifS were shown not to be essential to render active NifB, the lack of NifB-co was attributed to a lack of [Fe-S] cluster precursors to assemble NifB-co (and hence FeMo-co). It is important to note that NifB-co is a biosynthetic intermediate not only of FeMo-co but also of the FeV-co and the FeFe-co of alternative vanadium and iron-only nitrogenases. Consistently, nifU and nifS, mutants were shown to be defective in Mo-nitrogenase, V-nitrogenase and Fe-only nitrogenase activities [7].. !. $/!.

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his schematic model illustrates the enzymatic machinery (shown above) involved in different steps of nitrogenase metallocluster assembly (shown below). Early steps involve NifS and NifU for the assembly of NifH [4Fe-4S] cluster and the precursors of Pcluster and FeMo-co of NifDK. P-cluster biosynthesis ocurrs in situ by the NifHdependent condensation of the [4Fe-4S] pairs into the P-cluster. On the other hand, FeMo-co biosynthesis occurs outside NifDK. NifB catalyzes the first committed step of FeMo-co biosynthesis by assembling NifB-co, the central [FeS-C] core of FeMo-co. NifB-co is then transferred to the NifEN scaffold via NifX. !. $"!.

(36) ! ! Maturation to FeMo-co occurs within a putative NifEN/NifH complex by sequential addition of Fe, Mo and homocitrate. Finally, FeMo-co is transfered by NafY to apo-NifDK to generate active NifDK protein.. "#7#M!:+.DA!./,@!3+@C(%!NH%<LO!5('3*%/3!*,!*9%!5,/%!,.!H%I,<5,! ! The nifB gene product encodes a protein having a S-Adenosyl Methionine (SAM) radical motif CX3CX2C at its N-terminal region [72]. The Cterminal end of the protein comprises a NifX-like domain that is conserved in proteins with ability to bind FeMo-co and its biosynthetic precursors [10, 73]. As mentioned above, NifB participates in an early biosynthetic step that is common to FeMo-co, FeV-co and FeFe-co biosyntheses. Therefore, mutants lacking nifB were incapable of diazotrophic growth under all conditions tested [5, 9]. Consistently, regulation of nifB expression by the transcriptional activators of all three nitrogenases, NifA, VnfA and AnfA, has been reported [6]. NifB catalyzes the conversion of simple [2Fe-2S] or [4Fe-4S] clusters, donated by NifU, into a complex [Fe-S] cluster denominated as NifB-co in a reaction that involves radical chemistry [74]. Interestingly, the metabolic product of NifB, termed NifB-co, was purified and studied before purification of active NifB had been accomplished [75]. NifB-co comprises the central [(6-8)Fe-9S-C] core of FeMo-co but does neither contain a heterometal (e.g. Mo) nor homocitrate [75, 76]. Early experiments showed that NifB-co served as precursor to FeMo-co in the in vitro FeMo-co synthesis assay [75], and that it was the source of most (if not all) Fe and S present in FeMo-co [77]. The NifB protein was first purified from A. vinelandii cells [74]. Isolated NifB was a 110-kDa homodimer containing ca. 12 Fe atoms and exhibiting an UV-visible spectrum typical of [Fe-S] proteins. Changes in the NifB redox state and incubation with SAM altered the properties of its UV-visible spectrum, as expected for a redox-responsive SAM radical protein. Isolated NifB did neither carry NifB-co nor was readily active in supporting in vitro FeMo-co synthesis. !. $#!.

(37) ! ! However, after incubation with Fe2+ and S2-, Fe content increased to ca. 18 atoms and NifB became active [74] providing the first demonstration of complete in vitro FeMo-co synthesis from its atomic components. The work of Curatti also laid the groundwork for further mechanistic experiments by showing that radical chemistry was absolutely required for NifB activity. Later on, it was shown that the carbide atom at the center of FeMo-co had its origin in the methyl group of SAM [78]. An interesting observation is that NifB-co could be inserted in place of FeMo-co into nitrogenase in vitro. This artificial NifDK/NifB-co complex was capable of H+ and C2H4 reduction but not N2 fixation [79]. This observation emphasizes the importance in vivo of carrier proteins in redirecting metal precursors to the appropriate target proteins (see below).. "#7#P!HB0:A!)-!'-Q-,R-!/,(%!+-!:7!.+0)*+,-!! ! Electron donation processes are essential not only in the N2 reduction but also in many steps of FeMo-co biosynthesis. Electron donation involved in Mo-nitrogenase have been identified, in K. pneumonia, Azoarcus sp, and Rhodobacter capsulatus. In K. pneumoneiae NifF flavodoxin donates electrons to NifH, which in turn is the obligate electron donor to NifDK [80]. In R. capsulatus, an FdxN homolog is involved both in electron transfer to NifDK for N2 reduction, and in the biosynthesis of nitrogenase components [81, 82]. In Azoarcus sp. strain BH72, deletion of the fdxN homolog affected switch-off of nitrogenase activity [83] but no effect on nitrogenase activity nor biosynthesis was observed. Among the fifteen ferredoxins encoded in the A. vinelandii chromosome, FdxN can be classified as a type 2[4Fe-4S]. Thus class type includes ferredoxins. with. two. metal. cluster. binding. domains. containing. a. CysX2CysX2CysX3Cys and a CysX2CysX7~9CysX3CysX3~5Cys motifs, although the cysteine labeled in bold does not appear in the sequence of the A. vinelandii !. $$!.

(38) ! ! FdxN [1]. The fdxN gene is located into the minor nif cluster region downstream of nifB. Co-expression of nifB and fdxN occurs at similar levels under diazotrophic conditions that allow for the expression of either Mo or alternative nitrogenases [84]. Mutation in fdxN partially impaired in vivo nitrogenase activity and diazotrophic growth, indicating that although not essential, FdxN is involved in some aspect of nitrogenase activity, biosynthesis or regulation [9].. "#7#S!:+.TA!F+/%5*+-&!I,!*,!H%I,<5,!3?-*9%3+3! ! NifDK represents up to 5% of the total cellular protein accumulated under diazotrophic growth. Thus, N2-fixing A. vinelandii cells must cope with a large demand for Mo, a low abundance transition metal (1-2 ppm in soils). A. vinelandii produces siderophores, low-molecular-weight molecules with highaffinity for cation metals, to aid in Mo (and Fe) acquisition [85]. Unfortunately, siderophores can bind to other metals, such as W, which can eventually be incorporated into FeMo-co rendering inactive nitrogenase [86]. To discriminate against tungstate, A. vinelandii carries ATP Binding Cassette transport systems that are highly-specific for molybdate [87]. Three copies of the modABC operon are found in the A. vinelandii genome [1], as opposed to a single copy in the closely related bacterium Pseudomonas stutzeri. A. vinelandii has a unique Mo-accumulation system based on a Mo storage (MoSto) protein. MoSto is a $3%3 heterohexamer of the mosA and mosB gene products with capacity to store up to 100 Mo atoms [88] in the form of complexes of polynuclear oxoanions [89]. In addition, cellular systems are in place to keep Mo homeostasis and to direct molybdate to the corresponding Mo-dependent enzymes (Fig. 6). The molbindin ModG appears to be responsible for directing Mo to N2 assimilation pathways, such as nitrate reductase or nitrogenase [87]. NifO has been related to Mo balance between nitrate reductase and nitrogenase. It was suggested that NifO would direct Mo towards FeMo-co biosynthesis, thus impairing development of nitrate reductase activity [90].. !. $%!.

(39) ! ! The nifQ gene was identified by screening Nif- mutants, the phenotype of which could be reverted by a large excess of molybdate or cysteine in the growth medium [91, 92]. Although nifQ mutants did not accumulate molybdate they were neither impaired in molybdate transport nor in the activity of alternative nitrogenases [5, 9] nor Mo-co-containing enzymes such as nitrate reductase [59]. NifQ proteins are found in all diazotrophic species of the Proteobacteria phylum, with the exception of some Rhizobia. NifQ proteins do neither contain molbindin domains nor show sequence similarity to MosA or MosB. They do contain. a. highly. conserved. C-terminal. putative. metal-binding. motif. CX4CX2CX5C. As isolated from A. vinelandii, NifQ was a monomeric 20-kDa oxygensensitive protein containing ca. 3 Fe atoms and 0.30 Mo atoms per monomer. NifQ displayed a UV-visible spectrum typical of [Fe-S] proteins. Electronic Paramagnetic Resonance (EPR) and Electron spin echo (ESE)-EPR analyses revealed that NifQ carried a novel redox-responsive [Mo-3Fe-4S] cluster [93]. In. vitro. FeMo-co. synthesis. assays. with. purified. components. demonstrated that NifQ serves as unique Mo source for FeMo-co synthesis. Comparison of Mo-content in Nif proteins before and after the FeMo-co synthesis reaction revealed that, only in the presence of NifH, Mo was effectively mobilized from NifQ to NifEN, demonstrating that all three proteins were required for Mo transfer [93]. The exact reaction(s) carried out by NifQ are not known. The complete processing of Mo from molybdate (MoVI) to the state found in FeMo-co (MoIV) requires at least three chemical transformations: (i) replacement of O ligands by S ligands, (ii) reduction of Mo from MoVI to MoIV, and (iii) insertion into an [Fe-S] environment. It has been suggested that the role of NifQ could be related to some (or all) of these changes [94].. !. $&!.

(40) ! !. &. & & (478/9& :2& R5;L,E9>8M& ./06641^4>7& 65/& (9R5Q15& ,45-L>.@9-4-& 15MA/4-9-& 0.& ;90-.& 64=9& A/519--9-2 (i) molybdate harvesting by siderophores, (ii) molybdate transport and discrimination against tungstate, (iii) Mo accumulation and homeostasis, (iv) Mo sorting to the appropriate pathway, and (v) Mo insertion into FeMo-co. & & & & & !. $'!.

(41) ! !. "#7#U!:+.V!)-B!*9%!+-5,/C,/)*+,-!,.!9,@,5+*/)*%!+-*,!H%I,<5,! ! The nifV gene product is a homocitrate synthase that catalyzes the condensation of acetyl coenzyme A and 2-OG to render R-homocitrate [95]. nifV mutants exhibited slow diazotrophic growth rates [96], a phenotype that could be reverted in vivo by supplementing the growth medium with homocitrate [97]. V- and Fe-only nitrogenase dependent growth was also impaired in these mutants [7], indicating that homocitrate was part of FeV-co and FeFe-co as well. K. pneumoniae nifV mutants have been shown to incorporate citrate into a non-functional form of FeMo-co in vivo [98]. The situation was more complex in A. vinelandii where a mixture of organic acids replacing homocitrate in the cofactor was found [99]. In vitro FeMo-co synthesis assays carried out with analogous organic acids in place of homocitrate resulted in syntheses of cofactors with altered catalytic properties [100, 101]. It is not clear how the nitrogen-fixing cell manages to discriminate between homocitrate and other analogous. organic. acids. during. FeMo-co. biosynthesis.. Homocitrate. incorporation occurs within NifEN, presumably after Mo incorporation has taken place [102]. It is possible that discrimination occurs within the NifEN/NifH complex. It is also possible that homocitrate concentration in the cell was so high that it would preclude incorporation of other organic acids.. ! ! ! ! !. !. $(!.

(42) ! !. "#7#"W!:+.X:A!)!-,B%!+-!*9%!H%I,<5,!E+,3?-*9%*+5!C)*9R)?! ! NifEN is a 200-kDa $2%2 heterotetramer of the nifE and nifN gene products that carries two identical [4Fe-4S] clusters at the interface of both subunits [103]. If isolated from the appropriate genetic backgrounds, NifEN preparations exhibit trapped FeMo-co biosynthetic intermediates [104, 105]. NifEN is absolutely required for FeMo-co synthesis in vivo [3] and in vitro [106]. NifEN displays high similarity with NifDK at several levels, including amino acid sequence similarity, [107], position of their metal clusters within the protein [108] and the ability to catalyze C2H2 and azide reduction albeit at very low rates [109]. It was the amino acid sequence similarity of NifEN to NifDK that led to the proposal of NifEN acting as molecular scaffold for FeMo-co biosynthesis [107]. NifEN is the central node of the FeMo-co biosynthetic pathway, where Mo, homocitrate (and possibly additional Fe) are incorporated into NifB-co (Fig. 5) [51]. Briefly, NifB-co, is transferred from NifX to NifEN, where it is converted into the VK-cluster (named after Dr. Vinod K. Shah) [110]. Although both NifBco and the VK-cluster lack Mo and homocitrate and serve as precursors to FeMo-co, there are some differential properties that indicate they are not the same precursor. First, while NifB-co is EPR silent, the VK-cluster shows EPR signals both in reduced and oxidized states [110]. Second, EXAFS and NRVS analysis suggest that NifB-co is no larger than the central [6Fe-9S-C] core of FeMo-co [76], whereas the VK-cluster was proposed to be a larger [8Fe-9S] cluster [111]. The recently solved NifEN crystal structure confirmed the assignment of 8 Fe atoms for the VK-cluster. Third, NifEN-mediated Fe incorporation into NifB-co at capping positions external to the [6Fe-9S-C] core was achieved in vitro (Rubio laboratory unpublished results). In addition to the VK-cluster, NifEN purified from a "nifH background has been shown to contain Mo in a separate [Mo-3Fe-4S] cluster environment [112]. Occupancy levels for this cluster were low and dependent on the purification method used, probably due to cluster instability [105]. Nevertheless, it was !. $)!.

(43) ! ! shown to serve as Mo source during FeMo-co synthesis in vitro. The composition of this cluster resembles the one found in NifQ preparations and, since NifQ has been shown to be able to transfer Mo to NifEN in vitro [93], a logical proposal is that the [Mo-3Fe-4S] cluster within NifEN derives from the NifQ cluster. Another possibility is that this cluster represents a NifQindependent Mo insertion pathway that would operate with lower efficiency. This pathway would be responsible for the reversion of the nifQ mutant phenotype by the presence of 1000-fold molybdate into the growth medium [91, 113]. Interestingly, NifEN is able to substitute for the homologous VnfEN protein of the V-nitrogenase [114]. This finding raises questions regarding the specificity of NifEN in Mo insertion into FeMo-co and opens the possibility of other elements providing this specificity [94]. Finally, NifEN appears to be the site where homocitrate is incorporated into the cofactor in a reaction that requires NifH [102] .. !. !. $.!.

(44) ! !. "#7#""!:+.YA!*9%!-+*/,&%-)3%!@,,-(+&9*+-&!C/,*%+-!! ! NifH (also referred to as dinitrogenase reductase, Fe protein or nitrogenase component II) is the obligate electron donor to NifDK. NifH is a 60kDa homodimer of the nifH gene product. The NifH structure revealed a twofold symmetric enzyme with Mg2+!ATP binding sites located at the dimer interface within each monomer. A single [4Fe-4S] cluster is coordinated at the dimer interface through Cys97 and Cys132 of each NifH polipedtide chain [115]. NifH undergoes conformational changes during Mg2+!ATP binding and hydrolysis in a process following electron transfer from the [4Fe-4S] cluster of NifH to the Pcluster of the NifDK component [116]. Three accessory proteins are necessary to synthesize active NifH, namely NifU, NifS and NifM [4]. NifM is similar to prolyl isomerases and has been proposed to induce a conformational change on NifH that precedes incorporation of its [4Fe-4S] cluster [117]. NifU and NifS are involved in the assembly and delivery of the [4Fe-4S] cluster of NifH [70] (Fig. 5). NifH is a moonlighting protein with at least three essential roles in the nitrogenase system: (i) it is required for electron transfer to the NifDK component during catalysis, (ii) it is required to assemble P-clusters from pairs of [4Fe-4S] cluster precursors, and (iii) it is essential to FeMo-co biosynthesis, in which process it probably plays multiple roles. Not all of NifH capabilities are required for the performance of all its functions. Many lines of evidence show that Mg2+!ATP hydrolysis and electron transfer are required for catalysis but not for P-cluster nor FeMo-co biosynthesis. First, nifM mutants were shown unable to fix N2 but able to support FeMo-co biosynthesis [118]. Second, [4Fe-4S] cluster-deficient apoNifH (generated by chemical treatment of NifH to remove the metal clusters) was able to participate both in P-cluster synthesis and in FeMo-co synthesis [119]. Third, NifH variants with altered properties of Mg2+!ATP binding and/or hydrolysis could carry out FeMo-co biosynthesis [120, 121]. On the other hand, more recent experiments indicate that NifH must be able to hydrolyze !. %/!.

(45) ! ! Mg2+!ATP and to transfer electrons in order to be active in FeMo-co biosynthesis [111]. NifH is absolutely required for FeMo-co biosynthesis. Neither Mo nor homocitrate are incorporated into the cofactor in the absence of NifH. However, the exact mechanism by which NifH exerts its role remains unclear. NifEN and NifH are able to interact transiently with each other [121] and, in fact, Mo transfer from NifQ to NifEN occurs only in the presence of NifH [93]. It has been proposed that NifH would play its role of facilitating Mo insertion into the VKcluster simply by docking with NifEN and exerting some sort of conformational change on it [51]. The proposal of NifH being the element that selectively incorporates Mo into FeMo-co discriminating against other heterometals has long been discussed. Several observations do not support a role for NifH and other dinitrogenases reductases in specifying the heterometal to be inserted into the cofactor and point out to other proteins (e.g. NifQ) being potentially responsible for heterometal discrimination. VnfH, the equivalent protein in the Vnitrogenase, could replace NifH in FeMo-co biosynthesis [122]. Similarly, AnfH of the Fe-only nitrogenase supported FeMo-co synthesis in vivo [123]. Bishop and collaborators proved that NifH was able to support V-dependent diazotrophic growth in the absence of VnfH [124]. Finally, it is well known that NifH must be present to render homocitratecontaining FeMo-co [102, 111]. However, incorporation of homocitrate into an isolated Mo-containing FeMo-co precursor has not yet been reported. Thus, a direct role for NifH in homocitrate incorporation into FeMo-co precursor remains hypothetical.. !. %"!.

(46) ! !. "#7#"7!I%*)((,5('3*%/!5)//+%/!1%35,/*2!C/,*%+-3! ! Once a cofactor has been synthesized on a scaffold, it needs to be transferred to its target protein. When prosthetic groups are very labile and oxygen-sensitive, direct diffusion is unlikely and the metal clusters are expected to be always protein-bound within the cell [10]. Additionally, there is a rapid demand for nitrogenase synthesis during diazotrophic growth, which taken together, might explain the existence of proteins involved in metallocluster delivery. The nifX gene is clustered into a single operon together with nifEN. The nifENX gene cluster is in fact widespread among bacteria, suggesting the three gene products have a related role. NifX is a 17-kDa single-domain protein. Although unable to bind. 55. Fe or. 99. Mo [102, 125] or assemble an [Fe-S] cluster,. the product of the nifX gene has been shown able to bind FeMo-co and FeMoco precursors [110]. Early studies speculated with a role of NifX in the incorporation of homocitrate into a FeMo-co precursor [102], or as a negative regulator of nif-gene expression in response to NH4+ concentration and O2 [126]. However, recent in vitro experiments have demonstrated another roles for NifX [110]. First, it would work as donor of at least two FeMo-co precursors (NifB-co and VK-cluster) to NifEN. NifX and NifEN do not form a stable protein complex, but a transient interaction occurs for the metal cluster exchange to happen. Second, NifX would function as storage of FeMo-co precursors, redirecting labile metal clusters to NifEN. This might be especially relevant to buffer the flux of FeMo-co precursors under stress conditions, hence minimizing metal cluster losses. NifX-like domains are present in a group of nitrogenase-related proteins, and thus serves to define a family of nitrogenase cofactor binding proteins, including VnfX and the C-terminal domains of NifB, NifY, NafY and VnfY. NafY is probably the best characterized among them [10, 127, 128]. NafY is a 26-kDa protein with a double role in apo-NifDK stabilization and in FeMo-co insertion into apo-NifDK. Two functional domains can be defined in !. %#!.

(47) ! ! NafY, with each role mostly assigned to each domain. The 12-kDa N-terminal domain is sufficient to bind to apo-NifDK in the absence of the rest of the protein. NMR solution structure of the N-terminal domain of NafY revealed that it contained a sterile alpha motif domain, a structure frequently involved in protein-protein interactions [129]. This domain represented the first apo-NifDK binding structure known, other than NifH, and it exhibited a novel fold for apoNifDK binding, different from what is observed in the NifH structure [115]. Interestingly, excess of N-terminal NafY domain or full-length NafY had a negative effect on apo-NifDK reconstitution in vitro. The. 14-kDa. C-terminal. domain. was. shown. to. bind. FeMo-co. autonomously. The crystal structure of the core domain of NafY (defined as the C-terminal domain missing the last 13 amino acid residues) represents the only known FeMo-co binding fold different from that of NifDK [130]. Mutational analyses indicated direct implication of the His121 residue in FeMo-co binding [128]. These results suggest a model with a series of histidine residues involved in FeMo-co insertion into apo-NifDK. FeMo-co-bound to NafY via His121 would be donated to $-His362 (at the entrance of the insertion funnel in apo-NifDK), followed by the entry into the positively charged environment created by the His triad ($-His274, $-His442 and $-His451) and finally donation to $-His442, as one of the ligating residues of FeMo-co in NifDK [56]. Given the low affinity of NafY for NifB-co and the ability of apo-NifDK to bind NifB-co [79], this might be a physiological mechanism to couple FeMo-co biosynthesis to apo-NifDK activation, while preventing insertion of biosynthetic intermediates into the nitrogenase active site. NifX and NafY are not essential for in vitro FeMo-co synthesis or in vivo diazotrophic growth under standard laboratory conditions [10, 106]. However, several caveats need to be considered in order to appreciate their relevance. First, functional overlap among members of this family complicates the finding of a mutant phenotype in deletion mutants. A BLAST search for NifX-like sequences reveals two additional homologues in the genome of A. vinelandii [1], in addition to the above-mentioned members of this family. Thus, functional !. %$!.

(48) ! ! redundancy might obscure the observation of a phenotype in single mutant strains. Second, diazotrophic growth experiments of deletion mutant strains are typically carried out under optimal laboratory conditions. This might preclude the observation of phenotypes present under nutrient-limited environmental growth conditions. For instance, double "nifX "nafY mutant or triple "nafY "nifY nifX::kan mutant were impaired in diazotrophic growth under Mo starvation conditions [10]. Similar observations indicating the requirement of NifX under Fe-depleted conditions were reported in Herbaspirillum seropedicae [131]. Third, in vitro experiments proved the additive stimulatory effect of NifX and NafY on FeMo-co biosynthesis when present in the reaction mixture [106]. NifX and NafY were not required for NifB-co synthesis, but were able to independently increase apo-NifDK activation. Fourth, proteins involved in metal cluster storage and delivery have been described in other cofactor biosynthetic pathways, including the Mo-co carrier protein in Chlamydomonas reinhardtii [132], the IscA and ErpA carriers in E. coli [133], and the mammalian MMS19 protein for [Fe-S] cluster assembly [134], to name a few. Similarly, enzymespecific chaperones relevant to metal cofactor insertion into multi-subunit metalloenzymes have been reported in other systems, such as the NarJ chaperone from E. coli [135] and the copper superoxide dismutase from Saccharomyces cerevisiae [136]. Hence, it is not surprising to find proteins with similar roles in the FeMo-co biosynthetic pathway.. !. %%!.

(49) ! !. ! ! ! ! ! ! ! ! ! ! ! ! ! ! !. %&!.

(50) ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !. Chapter 2: Objectives ! ! ! ! ! ! ! !. !. %'!.

(51) ! ! The possibility to replace chemically-synthesized nitrogen fertilizers by a process of endogenous fixation of atmospheric N2 by major crop plants is a holy grail in plant biotechnology. Attempting to advance in such direction, our laboratory is trying to genetically transfer nitrogenase into non N2-fixing eukaryotic organisms. In order to implement the complex machinery required for nitrogenase biosynthesis in heterotopous hosts, it is important to acquire good knowledge about the roles of gene products involved, the intensity of gene expression, the timing of intervention and the in vivo stoichiometry of proteins involved in this process. In addition, some aspects of the biosynthesis of nitrogenase metal clusters, FeMo-co and the P-cluster, are still unclear. Elucidation of new roles and reaction mechanisms is important to determine whether or not their function could be replaced by housekeeping gene proteins in heterotopous hosts. The specific objectives of this thesis are: ". To determine Intracellular concentration of Nif proteins and nif gene. expression levels in cells of Azotobacter vinelandii along its adaptation to diazotrophic growth. " To elucidate the role of FdxN in the molybdenum nitrogenase. " To determinate the structure and function of molybdenum groups in NifQ and establish which of them are involved in molybdenum donation to FeMo-co biosynthesis.. ! ! ! !. %(!.

(52) ! ! ! ! ! ! !. !. Chapter 3: Results.. !. 3.1 Time-course analysis of nif mRNA and Nif protein accumulation upon derepression of nitrogenase in A. vinelandii. !. %)!.

(53) ! ! Activation of N2 fixation is a tightly regulated process, mainly because of the high-energy demand it imposes. Switching from N2-fixation repressing conditions to the N2 fixing state is a complex process that in the model aerobic free-living bacterium A. vinelandii affects expression of up to 400 genes [17]. In furtherance of providing a framework to engineer N2 fixation in nondiazotrophic organisms, we are interested in obtaining information about: - The intensity of nif gene expression along the time, defining at what moment expression starts, reaches a maximum and fades down to a new basal level, or starts another wave of expression. - The precise order in which each Nif protein intervenes during transition to diazotrophic growth and the stoichiometric balance amount different nif proteins, including the stage dominated by nitrogenase metal cofactor biosyntheses and the stage dominated by activity of mature nitrogenase. - The effect of mutations in regulatory, structural and biosynthetic genes on the balance of nif gene expression. - The connection of these data with nitrogenase activity registered in the cell. Following the methodology described in Fig. 7, we have quantified the accumulation of mRNA for 17 genes involved in N2 fixation, including 10 essential Mo-dependent N2 fixation genes (nifH, nifD, nifK, nifE, nifN, nifU, nifS, nifM, nifB and nifQ), 4 genes that code for non-essential proteins with assigned roles on N2 fixation (fdxN, clpX2, nifX, nafY), elements responsible of the regulation of nif gene expression (nifA, nifL), and a nif gene of unknown function in A. vinelandii (nifY). The research strategy includes analyzing the wild-type strain (DJ), a strain partially impaired in N2 fixation (!fdxN) [9], a strain incapable of nif gene expression (!nifA) [137], a strain fully defective in Monitrogenase activity because it lacks the MoFe protein structural genes ("nifDK) [138], and a strain unable to synthesize active site FeMo-cofactor (!nifB) [74]. In addition, the intracellular molar concentrations of NifH, NifD, NifK, NifE, NifN, NifU, NifS, NifX, NifY, and NafY were established by using. !. %.!.

(54) ! ! quantitative immunoblot analysis of whole cells and estimating the total cell volume in the samples (Fig. 8). A version of this section has been published: Poza-Carrion, C., JimenezVicente, E., Navarro-Rodriguez, M., Echavarri-Erasun, C. & Rubio, L. M. (2014) Kinetics of Nif gene expression in a nitrogen-fixing bacterium, J. Bacteriol. 196, 595-603.. & & &. !. &/!.

(55) ! !. !"#$%&'()&*+,-"./*+'/-". <=>". 01233". 4*"5(5/" *(+'/6&*,7&" ,.85(+9". .1=>". AB/-&" .&--7". ?@:<". A&7+&'*" C-/+". #$%'&77(/*" !"#"6&*&7". >..;);-,8/*" =(D"%'/+&(*7". :&--"5/-;)&*". (478/9&O2&R9.@5E5;57L&9MA;5L9E&.5&0>0;LZ9&!"#&9HA/9--45>&8A5>&>4./579>& -.9AQE5U>2&NH4+-grown cells (green) were collected by centrifugation, washed with NH4-free (yellow) or NH4+-containing (green) medium (control cultures), and resuspended in the same medium at a final OD600 of 0.5. Cells were then incubated at 30°C with shaking (200 rpm) in 9 independent Erlenmeyer flask. At 0, 10, 30, 60, 120, 180, 240 and 420 minutes each one of these flasks was collected and subjected to the following analyses: - Determination of in vivo C2H2 reduction activity - Cell volume determination - OD600 measurement - nif mRNA quantification by qPCR - Quantitative immunoblot analysis & & &. !. &"!.

(56) ! !. !m3/ml. 109. 108. 0. 1. 2. 3. 4. Time (h). 5. 6. 7 &. & & & & (478/9&<2 _0/40.45>&56&.5.0;&19;;&=5;8M9&4>&0>&$1-."!+/)!0""&18;.8/9&E8/4>7&.@9& -U4.1@& .5& E40Z5./5A@41& 7/5U.@& 15>E4.45>-2 Total cell volume was estimated by measuring average cell volume and multiplying this value by the number of CFU per ml of culture.. !. &#!.

(57) ! !. 3.1.1 Diazotrophic growth and in vivo C2H2 reduction activities of A. vinelandii wild-type, !fdxn, !nifA, !nifB and !nifDK strains ! As a result of their inability to fix N2 using the Mo-nitrogenase, !nifA, !nifB and !nifDK strains were unable of grow under standard diazotrophic conditions (data not shown). On the other hand, the "fdxN strain exhibited a Nif+ phenotype, although it diazotrophic growth rate was severely affected (see section 3.2.1). In vivo C2H2 reduction activities of A. vinelandii wild type, !fdxn, !nifA, !nifB and !nifDK mutants under diazotrophic growth conditions were determined (Fig. 9). Cultures were growth in Burk media lacking NH4+ in order to initiate Mo-nitrogenase synthesis. C2H2 reduction activity by nitrogenase starts one hour after induction, and in the wild-type strain reaches maximum values four hours after derepression. C2H2 reducing activities of the !nifA, !nifB and !nifDK mutant strains were unable to reduced C2H2 under Mo-nitrogenase derepressing conditions (as expected), whereas the !fdxN mutant strain was severely affected in nitrogenase activity, showing 30% of wild type levels.. !. !. &$!.

(58) ! ! !. ! ! ! Figure 9. In vivo C2H2 reduction activities of A. vinelandii wild type and the !fdxN, !nifB, !nifA and !nifDK mutants. !. !. &%!.

(59) ! !. 3.1.2. Time-course. of. nif. gene. expression. and. Nif. protein. accumulation in wild-type cells upon nitrogen step-down.. !"#&MG*"&0118M8;0.45>&;9=9;-&4>&$1-."!+/)!0""-U4;EQ.LA9&19;;-&8A5>& 9>.9/4>7&E40Z5./5A@41&7/5U.@& ! Absolute levels of nif-specific mRNAs accumulation were estimated by quantitative real-time PCR (qRT-PCR) against the results using known amounts of synthetic DNA amplicons as template. nif gene mRNA levels were then compared to 16S rRNA levels present in each sample to yield normalized absolute signals (NAS). Fig. 9 and Fig. 10 show that A. vinelandii responded rapidly to nitrogen step-down by expressing nif genes and developing nitrogenase activity. The nifLA operon was the fastest to respond with nifA exhibiting a narrow pulse of expression that peaked 30 min after NH4+ removal from the medium. Expression of genes within the iscAnifnifUSVcysE1nifnifWZM clpX2 cluster also peaked after 30 minutes exhibiting NAS values for nifU of (10), nifS (7.3), nifV (3.6), and clpX2 (1.8). Most genes involved in FeMo-co biosynthesis (nifENX and nifB fdxN nifOQ operons) showed maximum mRNA levels 1 h after derepression with NAS values ranging from 2 to 7. Similarly, nitrogenase structural genes nifH, nifD and nifK showed maximum mRNA levels 1 h after derepression but NAS values ranged from 30 to 50. In contrast, nifY showed the slowest response with accumulation peak at 2 h and a NAS of 4.3. Two main patterns of mRNA accumulation occurred when nitrogenase activity was at steady state: while nifH, nifD, nifK, nifE, nifN, nifX and nafY levels were at least 25% of their maximum NAS 7 h after derepression, all other genes returned to basal NAS levels.. !. &&!.

(60) ! !. !. &'!.

(61) ! !. Figure 10. Time-dependent profile of nif gene induction in the wild-type strain of A. vinelandii. (A to C) Expression levels of genes within the major nif cluster, which contains nifHDKTYENX iscAnif nifUSV cysE1nif nifWZM clpX2 nifF. (D to F) Expression levels of genes within the minor nif cluster, which contains rnfABCDGEH nafY in one DNA orientation and nifLAB fdxN nifOQ rhdN grx5nif in the opposite DNA orientation. Black arrows in ORF maps indicate predicted "54-dependent promoter regions. The gray arrow indicates a hypothetical constitutive promoter region. Dot lines represent unstudied interspersed genes within each operon. Data is the average of at least three biological replicates ± SE.. & ! ! ! ! ! !. &(!.