Protein Crystallography Reveals a Role for the FS0 Cluster of Escherichia coli Nitrate Reductase A (NarGHI) in Enzyme Maturation

Abstract

We have used site-directed mutagenesis, EPR spectroscopy, redox potentiometry, and protein crystallography to monitor assembly of the FS0 [4Fe-4S] cluster and molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). Cys and Ser mutants of NarG-His 49 both lack catalytic activity, with only the former assembling FS0 and Mo-bisPGD. Importantly, both prosthetic groups are absent in the NarG-H49S mutant. EPR spectroscopy of the Cys mutant reveals that the E m value of the FS0 cluster is decreased by at least 500 mV, preventing its participation in electron transfer to the MobisPGD cofactor. To demonstrate that decreasing the FS0 cluster E m results in decreased enzyme activity, we mutated a critical Arg residue (NarG-Arg 94 ) in the vicinity of FS0 to a Ser residue. In this case, the E m of FS0 is decreased by 115 mV, with a concomitant decrease in enzyme turnover to ϳ30% of the wild type. Analysis of the structure of the NarG-H49S mutant reveals two important aspects of NarGHI maturation: (i) apomolybdoNarGHI is able to bind GDP moieties at their respective P and Q sites in the absence of the Mo-bisPGD cofactor, and (ii) a critical segment of residues in NarG, 49 HGVNCTG 55 , must be correctly positioned to ensure holoenzyme maturation.Escherichia coli, when grown anaerobically with nitrate as respiratory oxidant, develops a respiratory chain terminated by a membrane-bound quinol:nitrate oxidoreductase (NarGHI) (1-3). This enzyme is an archetype of the complex iron-sulfur molybdoenzyme (CISM) 2 family (4) that includes E. coli formate dehydrogenase N (FdnGHI (5)), E. coli Me 2 SO reductase (DmsABC (6)), Wolinella succinogenes polysulfide reductase (PsrABC (7, 8)), Salmonella typhymurium thiosulfate reductase (PhsABC (9)), and Salmonella enterica tetrathionate reductase (TtrABC (10)). These enzymes and their close relatives contribute significantly to the remarkable metabolic diversity of bacteria.NarGHI and the other CISM archetypes comprise a mononuclear molybdenum cofactor (molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD))-containing catalytic subunit, e.g. NarG; a four-cluster protein subunit, e.g. NarH; and a membrane anchor protein, e.g. NarI. Each catalytic subunit contains a [4Fe-4S] cluster in addition to MobisPGD, and many of the membrane anchor subunits are also diheme cytochromes b. The five [Fe-S] clusters are referred to as FS0 -FS4, with increasing distance from Mo-bisPGD, and the entire electron transfer relay (ETR) in NarGHI, including the two hemes, spans a distance of almost 100 Å (2).In NarGHI, Mo-bisPGD provides the site of nitrate reduction at its redox-active molybdenum atom. The molybdenum atom is coordinated by two pyranopterin guanine dinucleotide (PGD) moieties via a bis-dithiolene linkage (11-13). These PGD groups are referred to as the P-and Q-pterins, and they are proximal and distal, respectively, to the FS0 [4Fe-4S] cluster. The bis-dithiolene coordination is supplemented by one oxo gr...

Q-Site Occupancy Defines Heme Heterogeneity in Escherichia coli Nitrate Reductase A (NarGHI)

“…14 The cardiolipin headgroup binds to a region within the complex where all three subunits converge and is stabilized by the conserved residues NarG-Tyr9, NarG-Arg6, NarH-Arg218, and NarI-Tyr28. Given that maturation of NarGHI is highly coordinated and involves a redox enzyme maturation protein (NarJ) and sequential insertion of its prosthetic groups, 13,37 it is unlikely that a significant proportion of the mature enzyme would be assembled without the structurally important cardiolipin. It is also unlikely that another anionic phospholipid (phosphatidylglycerol) binds in its place because the S330 strain is defective in phosphatidyl-glycerol and cardiolipin biosynthesis (pgsA null).…”

“…CIHR Author Manuscript CIHR Author Manuscript groups, 13,37 it is unlikely that a significant proportion of the mature enzyme would be assembled without the structurally important cardiolipin. It is also unlikely that another anionic phospholipid (phosphatidylglycerol) binds in its place because the S330 strain is defective in phosphatidyl-glycerol and cardiolipin biosynthesis (pgsA null).…”

Correct Assembly of Iron-Sulfur Cluster FS0 into Escherichia coli Dimethyl Sulfoxide Reductase (DmsABC) Is a Prerequisite for Molybdenum Cofactor Insertion

“…In the case of another Type II enzyme, E. coli NarGHI, the FS0 spectrum was observed at low temperature at g ϳ 5.0 (16,17). Further studies revealed that FS0 insertion was closely linked to that of the Mo-bisPGD cofactor (18). An important distinction between the Type II Cys group sequence of DmsA and that of NarG is that there is a His in the C A position of the latter and a Cys at this position in the former.…”

“…In the case of DmsABC, the system-specific chaperone DmsD plays a role in targeting DmsAB to the tat translocon and may have additional roles in maturation (26). We have recently demonstrated by protein crystallography that, in NarGHI, FS0 assembly is a prerequisite for Mo-bisPGD insertion (18). It is therefore of great interest to see if a similar sequence of cluster assembly and cofactor insertion occurs in the maturation of DmsABC.…”

“…We introduced a Type I Cys group into DmsA in two stages. We changed its sequence from 18 C A TVNC B GSRC C P 27 to 18 C A TYC B GVGC C G 26 (similar to that of formate dehydrogenase (FdnG)) and demonstrated that this eliminated both Mo-bisPGD binding and EPRdetectable FS0. We then combined this change with a DmsA R61K mutation and demonstrated that this additional change partially rescued Mo-bisPGD insertion.…”