title: Structural and Functional Studies of c-type Cytochromes from Anaerobic Ammonium-Oxidizing Bacteria
Creators: name: Akram, Mohd
Publisher: name: Heidelberg University Library
Year: 2019

description: Anaerobic ammonium oxidation (anammox) is a microbial process in the Earth's nitrogen cycle with both major ecological and technical significance. The anammox process is carried out by unique, slowly growing bacteria belonging to the phylum of Planctomycetes. These bacteria are indeed estimated to contribute to almost 50 % of dinitrogen (N2) release from the oceans into the atmosphere (Arrigo 2005; Lam 2011) and are increasingly being applied in environmentally friendly waste water treatment procedures. The cells of anammox bacteria are characterized by the presence of a large intracellular compartment called "anammoxosome" which is the location of their unique metabolic pathway. Anammox bacteria use the oxidation power of nitrite (NO2-) to anaerobically oxidize ammonium (NH4+) to dinitrogen gas and water via two highly unusual intermediates namely nitric oxide (NO) and hydrazine (N2H4). The final step of this metabolic pathway, the oxidation of hydrazine to N2, is catalyzed by an octaheme c-type cytochrome called hydrazine dehydrogenase (HDH). The electron transport between the various enzyme complexes involved in this metabolism is believed to be carried out by small c-type cytochromes, numerously present in anammox bacteria.

In this thesis work, the molecular structures of HDH and several small c-type cytochromes were determined by X-ray crystallography in order to elucidate their mechanism of action. Moreover, these proteins were also characterized by various biophysical and biochemical methods. The first part of this work (chapter 3) describes the investigation of hydrazine dehydrogenase (HDH). This enzyme is related to octaheme hydroxylamine oxidoreductases (HAO) and forms covalently crosslinked trimers (α3) which were previously shown to associate into higher oligomers in solution namely 24mers (α3)8 and 30mers (α3)10 with molecular masses of 1.6 and 2.0 MDa, respectively (Maalcke 2016). In the current study, the crystal structure of a 24meric hydrazine dehydrogenase complex purified from the anammox organism Kuenenia stuttgartiensis (KsHDH) was determined at 2.8 Å resolution. This intriguing complex is composed of eight conically shaped HDH trimers that are positioned at the corners of a cube pointing their apexes outwards. This assembly enables the interaction of 24 c-type hemes from each trimer to form an astonishing network of 192 hemes. In addition, 12 molecules of an unanticipated ~10 kDa protein identified as the gene product of kustc1130 were found at each edge of the cubic assembly between two neighboring HDH trimers. This complex has an overall molecular mass of 1.7 MDa.

Interestingly, an additional covalent crosslink between a conserved cysteine residue and the active site heme P460 was discovered which might contributed to the specificity of HDH towards hydrazine. This work was complemented by structural studies of both 24mer and 30mer HDH assemblies by cryo-electron microscopy (cryo-EM) by Dr. Kristian Parey. The KsHDH 24mer crystal structure could be well superimposed with the molecular model of an HDH 24mer determined by cryo-EM, which was, however, lacking the small binding partner protein Kustc1130. Various biophysical methods showed that oligomerization of HDH trimers to both 24mers and 30mers was promoted at high ionic strength (&gt;100 mM). Moreover, the presence of Kustc1130 preferably induced the formation of the 24mer assembly independent of ionic strength. HDH was found to be most active with its binding partner at 100-150 mM ionic strength. Therefore, it can be proposed that the 24mer HDH assembly with 12 molecules of its binding partner is the physiologically relevant oligomeric state of HDH.
The next part (chapter 4) describes the investigation of small c-type cytochromes namely the KsNaxLS complex and the monoheme cytochrome c Kustc0563 and its paralogue Kustc0562. KsNaxL and KsNaxS are the gene products of kusta0087 and kusta0088, respectively, and were purified as a stable complex from K. stuttgartiensis. The X-ray structure of the KsNaxLS complex was determined at 1.7 Å resolution and showed a single heterodimeric assembly in the asymmetric unit. The molecular model of KsNaxL shows a four-helix bundle fold typical for class II cytochromes c, whereas KsNaxS displays a typical class I cytochrome c structure. Importantly, KsNaxLS represents the first structure of a complex between class I and II monoheme c-type cytochromes. The heme iron in each subunit is coordinated by a rare cysteine ligand at its distal side. Biophysical investigations of KsNaxLS resulted in a molecular mass of approximately 24 kDa which is consistent with a heterodimer in solution. Moreover, the complex could be reconstituted in vitro from its individual subunits that were heterologously expressed in Shewanella oneidensis MR-1. UV-Vis spectroscopy revealed that the complex and its components possess a Soret band maximum at around 420 nm showing a unique blue shift upon reduction. Further UV-Vis spectroscopic analyses indicated binding of nitric oxide (NO) and carbon monoxide (CO) to the hemes in the KsNaxLS complex and its individual subunits. The obtained spectroscopic features match with those of CO-sensing as well as NO-scavenging and -shuttling hemoproteins. Finally, a pull-down assay indicated that KsNaxLS might interact with the hydrazine synthase (HZS) complex. Based on these results, one can propose a role of KsNaxLS as an NO-scavenger which binds free nitric oxide in the anammoxosome which would otherwise inhibit the activity of HDH. In addition, NaxLS might shuttle NO to HZS which uses this compound as a substrate.

The highly expressed class I monoheme c-type cytochrome Kustc0563 was the very first protein purified from the anammox bacterium K. stuttgartiensis (Cirpus 2005). In this study, the X-ray structure of heterologously expressed Kustc0563 was determined at 1.9 Å resolution. Biochemical studies showed that this protein can act as redox partner in hydroxylamine oxidation assays by hydroxylamine oxidase (HOX) and also in hydrazine oxidation by HDH. Interestingly, hydroxylamine oxidation activity was 6-7 times higher with Kustc0563 than with bovine cytochrome c as redox partner. On the contrary, Kustc0562, a paralogue of Kustc0563 possessing a similar redox potential, did not show redox activity with HOX, but displayed comparable activity to Kustc0563 in assays with HDH. Although the 3.3 Å resolution crystal structure of Kustc0562 was highly similar to Kustc0563, it revealed highly diverse surface electrostatics which might explain the different redox activities of the two proteins. 

Eventually, chapter 5 describes the purification and characterization of anammox proteins from granular sludge collected from a DEamMONification (DEMON®) reactor at a local waste water treatment plant. Phylogenetic analyses showed that the sampled biomass contained Brocadia fulgida as the sole representative anammox species. In addition, aerobic ammonium-oxidizing bacteria (AOB), represented by the genus Nitrosomonas could also be detected. Key metabolic enzymes from the anammox bacterium B. fulgida such as HZS, HDH as well as HOX could be purified from the biomass using anion exchange-, hydroxyapatite- and gel filtration chromatography. The identity of the isolated proteins was confirmed by mass spectrometry. All purified proteins showed characteristic UV-Vis spectra in their as-isolated, reduced and substrate-bound states. HDH and HOX were shown to be optimally active and were of sufficient quality for crystallization. Finally, the 3.0 Å resolution X-ray structure of B. fulgida HOX was determined which shows high similarity to K. stuttgartiensis HOX. This work proves the possibility of using granular sludge from large scale anammox reactors at waste water treatment plants as a convenient source for the purification of anammox proteins as well as their biochemical, biophysical and structural characterization.