Nitrogen is an essential element for all organisms. The most abundant form of nitrogen is atmospheric N2, which must first be fixed using either an abiotic or a biological energy-intensive process before it is bioavailable. Unfortunately, almost half of the fixed nitrogen that reaches agricultural lands as fertilizer is lost through biological and physical processes [[1], [2], [3], [4]]. Many soil microorganisms readily oxidize ammonium (NH4+) either via the comammox (complete ammonia oxidation) or nitrification pathways to nitrite (NO2−) and nitrate (NO3−), both of which can easily leach from soils and contribute to water pollution and devastating algal blooms (Fig. 1). NO3− and NO2− can also go through denitrification and be lost from the soil via the formation of N2 or the gaseous intermediates NO and N2O. Furthermore, fixed nitrogen can be lost as N2 when NH4+ and NO2− react in a comproportionating process known as anammox (anaerobic ammonia oxidation).

The Dissimilatory Nitrate Reduction to Ammonium (DNRA) pathway helps retain fixed nitrogen in the soil [[6], [7], [8], [9], [10], [11], [12]]. In this pathway, NO3− is reduced to NO2− in the first step and then further reduced to NH4+ in the second step, bypassing the processes of denitrification and nitrogen fixation [13]. The pentaheme enzyme cytochrome c nitrite reductase, NrfA, is responsible for this second step in an impressive six-electron reduction that occurs at a single active site [8,14,15]. The soluble, periplasmic enzyme reaches in vitro specific activities of over 1000 μmol NO2− min−1 mg−1 depending on the electron donor [[16], [17], [18], [19]].

There are two main types of NrfA-containing operons found in nature: nrfHA and nrfABCDEFG (Fig. 2). NrfH and NrfB are the redox partners to NrfA in their respective systems [20]. In the first system, NrfH transfers electrons from the membrane quinol pool to NrfA while in the second system, a NrfCD complex transfers electrons from the quinol pool to NrfB, which then shuttles the electrons to NrfA [21].

Although elucidating the architecture of the Nrf complexes has increased our understanding of electron movement during the DNRA reaction, the nature of the active complex and the physiological relevance of these protein-protein interactions remain largely unknown. The only crystal structure of a NrfAH complex to date, from Desulfovibrio vulgaris, reveals an unexpected [NrfA]:[NrfH] ratio of 2:1, resulting in NrfH binding asymmetrically at the NrfA-NrfA dimer interface. It is unknown if this binding arrangement can be extended to other organisms, but if this structure is physiologically relevant, then NrfH can only deliver electrons to one of the two NrfA enzymes. Dynamic light scattering data with purified NrfA, however, indicates that the enzyme may function as a monomer rather than a dimer in some organisms [16,22], although it is also possible that NrfA requires contact with its redox partner to form a stable, functional NrfA dimer. Furthermore, the storage and movement of electrons by NrfA and its corresponding redox partners are critical to the function of these complexes, but the mechanism by which electron transfer occurs and is regulated is still not fully understood. This article reviews the latest findings on the coordinated flow of electrons in NrfA, the proposed mechanism of the intriguing reaction that this enzyme catalyzes, and the structure-function relationships of NrfA.