NO Reductase from Bacillus azotoformans Is a Bifunctional Enzyme Accepting Electrons from Menaquinol and a Specific Endogenous Membrane-Bound Cytochrome c₅₅₁

Abstract

Bacillus azotoformans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique menaquinol-dependent nitric oxide reductase (qCu(A)NOR) with a Cu(A) center in its small subunit. The qCu(A)NOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the E(m) for all is close to 150 mV. Only one of these c cytochromes, which exhibits an alpha-band maximum at 551 nm, acts as a direct electron donor to qCu(A)NOR. Further investigation demonstrated that this cytochrome c(551) possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c(551) is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCu(A)NOR. The ability of qCu(A)NOR to accept electrons from both menaquinol and cytochrome c(551) might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus denitrificans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c(551), is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b(6)f complex serving the bioenergetic needs of the organism.

Spectroscopic characterization of heme iron–nitrosyl species and their role in NO reductase mechanisms in diiron proteins

“…Putative mechanisms of NO reductase. Resonance Raman characterization of heme/non-heme μ-oxo bridged diiron(III) cluster in oxidized cNOR (in 16 OH 2 (blue), 18 OH 2 (green) and difference spectrum (red)), and in a synthetic model in CH 3 CN with 20% water (in 16 OH 2 (blue), 18 OH 2 (green) and difference spectrum (red)). The ν as (Fe-O-Fe) modes are identified by their 40 cm −1 downshift upon exchange of the O-atom with 18 O-labeled water.…”

Nitrous oxide production and consumption: regulation of gene expression by gas-sensitive transcription factors

“…An unusual hybrid NOR has been described in the Gram-positive organism Bacillus azotoformans [12,13]. This is a two subunit enzyme that functions as a menaquinol : NO oxidoreductase, but can also accept electrons from small membrane-bound c-type cytochromes [13].…”

Antibacterial Screening of Soil Bacteria Isolates from Guimaras Island, Philippines against Escherichia Coli and Staphylococcus Aureus

“…Bacteria isolate I1 is a diplobacilli while others are either clustered or occurred individually. Among the different microorganisms inhabiting the soil, bacteria are the most abundant and predominant organisms [24]. Morphologically, soil bacteria are divided into three groups viz cocci (round/spherical), (rod-shaped) and spirilla/spirillum (cells with long wavy chains) [24].…”

“…Figure 5 shows the mean zone of inhibition of highland soil bacterial isolates against E. coli BIOTECH 1634 after 24 Figure 7 shows the mean zone of inhibition of lowland soil bacterial isolates against E. coli BIOTECH 1634 after 24, 48 and 72 hours of incubation. Bacteria isolates L1 (M=23mm, SD=10.77), L4 (M= 30.89mm, SD=4.88), and L5 (M=34.78mm, SD=3.60) have the highest zones of inhibitions and described as partially active after 24, 48 and 72 hours of incubation, respectively.…”