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Nitric oxide is an important molecule in all domains of life with significant biological functions in both pro- and eukaryotes. Anaerobic ammonium-oxidizing (anammox) bacteria that contribute substantially to the release of fixed nitrogen into the atmosphere use the oxidizing power of NO to activate inert ammonium into hydrazine (N2H4). Here, we describe an enzyme from the anammox bacterium Kuenenia stuttgartiensis that uses a novel pathway to make NO from hydroxylamine. This new enzyme is related to octaheme hydroxylamine oxidoreductase, a key protein in aerobic ammonium-oxidizing bacteria. By a multiphasic approach including the determination of the crystal structure of the K. stuttgartiensis enzyme at 1.8 Å resolution and refinement and reassessment of the hydroxylamine oxidoreductase structure from Nitrosomonas europaea, both in the presence and absence of their substrates, we propose a model for NO formation by the K. stuttgartiensis enzyme. Our results expand the understanding of the functions that the widespread family of octaheme proteins have.

Background: SoxAX enzymes initiate microbial oxidation of reduced inorganic sulfur compounds. Their catalytic mechanism is unknown., Results: Cyanide displaces the CysS(-) ligand to the active site heme following reduction by S(2)O(4)(2-) but not Eu(II)., Conclusion: An active site heme ligand becomes labile on exposure to substrate analogs., Significance: Elucidation of SoxAX mechanism is necessary to understand a widespread pathway for sulfur compound oxidation. SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E(m)) at pH 7.0 of approximately +210, -340, and -400 mV for the His/Met, His/Cys(-), and active site His/CysS(-)-ligated heme, respectively. Exposing SoxAX to S(2)O(4)(2-), a substrate analog with E(m) ~-450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E(m) ~-1140 mV), allows cyanide to displace the cysteine persulfide (CysS(-)) ligand to the active site heme. This provides the first evidence for the dissociation of CysS(-) that has been proposed as a key event in SoxAX catalysis.

Little is known about enzymatic quinone-quinol interconversions in the lipid membrane when compared with our knowledge of substrate transformations by globular enzymes. Here, the smallest example of a quinol dehydrogenase in nature, CymA, has been studied. CymA is a monotopic membrane tetraheme c-type cytochrome belonging to the NapC/NirT family and central to anaerobic respiration in Shewanella sp. Using protein-film electrochemistry, it is shown that vesicle-bound menaquinone-7 is not only a substrate for this enzyme but is also required as a cofactor when converting other quinones. Here, we propose that the high concentration of quinones in the membrane negates the evolutionary pressure to create a high affinity active site. However, the instability and reactivity of reaction intermediate, semiquinone, might require a cofactor that functions to minimize damaging side reactions.

Heme, a physiologically crucial form of iron, is a cofactor for a very wide range of proteins and enzymes. These include DNA regulatory proteins in which heme is a sensor to which an analyte molecule binds, effecting a change in the DNA binding affinity of the regulator. Given that heme, and more generally iron, must be carefully regulated, it is surprising that there are no examples yet in bacteria in which heme itself is sensed directly by a reversibly binding DNA regulatory protein. Here we show that the Rhizobium leguminosarum global iron regulatory protein Irr, which has many homologues within the alpha-proteobacteria and is a member of the Fur superfamily, binds heme, resulting in a dramatic decrease in affinity between the protein and its cognate, regulatory DNA operator sequence. Spectroscopic studies of wild-type and mutant Irr showed that the principal (but not only) heme-binding site is at a conserved HXH motif, whose substitution led to loss of DNA binding in vitro and of regulatory function in vivo. The R. leguminosarum Irr behaves very differently to the Irr of Bradyrhizobium japonicum, which is rapidly degraded in vivo by an unknown mechanism in conditions of elevated iron or heme, but whose DNA binding affinity in vitro does not respond to heme.