Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

Abstract

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (I) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at E m = ?197 mV (heme c) and -4.5 mV (heme b). Variable-temperature (4-120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe-4S]? cluster and overlapping signals associated with at least three types of ? centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called ''lowpH'' and ''high-pH,'' changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membranebound nitrate reductases.

Nitrate reduction associated with respiration in Sinorhizobium meliloti 2011 is performed by a membrane-bound molybdoenzyme

“…In contrast, Sm NR showed a high substrate affinity in line with NarGHI from Clostridium perfringens (K m = 100 lM) (SekiChiba and Ishimoto 1977) and Pyrobaculum aerophilum (K m = 58 lM) (Afshar et al 2001). The turnover number for Sm NR was found to be k cat = 12.1 ± 0.6 s -1 , similar to that found for NarGHI from Marinobacter hydrocarbonoclasticus 617 (Correia et al 2008).…”

“…K m values in the range 250-4000 lM have been reported for NarGHI enzymes from several prokaryote organisms (Forget 1974;Satoh 1981;Carlson et al 1982;Craske and Ferguson 1986;Blümle and Zumft 1991;Correia et al 2008). In contrast, Sm NR showed a high substrate affinity in line with NarGHI from Clostridium perfringens (K m = 100 lM) (SekiChiba and Ishimoto 1977) and Pyrobaculum aerophilum (K m = 58 lM) (Afshar et al 2001).…”

“…The same procedure performed in denaturating conditions failed to show heme dependent peroxidatic activity. This result suggests that this cofactor is not covalently bound to the protein and consequently would correspond to a b-type heme cofactor, as found in NarGHI proteins (Correia et al 2008). …”

The Mononuclear Molybdenum Enzymes

“…455 Nar’s typically exhibit two pH-dependent EPR signals, where a “low pH” signal ( g 1,2,3 = 2.001, 1.986, 1.964) distinguishes itself from the “high-pH” signal ( g 1,2,3 = 1.987, 1.981, 1.962) in the magnitude of the observed hyperfine splitting, a av = 9.6 G for the low-pH form and a av = 3.4 G for the high-pH form; in both signals, the protons are solvent-exchangeable. 455d It has been suggested that only the low-pH form is catalytically competent, 455e but more recent studies suggest that both forms may be important in catalysis. 452 A detailed investigation with the M. hydrocarbonoclasticus 617 Nar has demonstrated the formation of both high- and low-pH forms in the as-isolated enzyme.…”

Reductive activation of E. coli respiratory nitrate reductase

“…They have been purified and characterized from different organisms: Escherichia coli (Ec) [8], Paracoccus pantotrophus (Pp) [9], Haloarcula marismortui [10], and Marinobacter hydrocarbonoclasticus [11]. The prototypical Nar, Ec NarGHI consists of three subunits: NarG bears the Mo active site for nitrate reduction and a [4Fe-4S] cluster, NarH bears three [4Fe-4S] clusters and a [3Fe-4S] cluster, and NarI is attached to the membrane and bears two b-type hemes that oxidize quinols [12,13].…”