Heme P460: A (Cross) Link to Nitric Oxide

Abstract

Conspectus Ammonia-oxidizing bacteria (AOB) convert ammonia (NH3) to nitrite (NO2 –) as their primary metabolism and thus provide a blueprint for the use of NH3 as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NH2OH) to NO2 –. HAO uses the heme P460 cofactor as the site of catalysis. This heme is supported by seven other c hemes in each monomer that mediate electron transfer. Heme P460 cofactors are c-heme-based cofactors that have atypical protein cross-links between the peptide backbone and the porphyrin macrocycle. This cofactor has been observed in both the HAO and cytochrome (cyt) P460 protein families. However, there are differences; specifically, HAO uses a single tyrosine residue to form two covalent attachments to the macrocycle whereas cyt P460 uses a lysine residue to form one. In Nitrosomonas europaea, which expresses both HAO and cyt P460, these enzymes achieve the oxidation of NH2OH and were both originally reported to produce NO2 –. Each can inspire means to effect controlled release of chemical energy. Spectroscopically studying the P460 cofactors of HAO is complicated by the 21 non-P460 heme cofactors, which obscure the active site. However, monoheme cyt P460 is more approachable biochemically and spectroscopically. Thus, we have used cyt P460 to study biological NH2OH oxidation. Under aerobic conditions substoichiometric production of NO2 – was observed along with production of nitrous oxide (N2O). Under anaerobic conditions, however, N2O was the exclusive product of NH2OH oxidation. We have advanced our understanding of the mechanism of this enzyme and have showed that a key intermediate is a ferric nitrosyl that can dissociate the bound nitric oxide (NO) molecule and react with O2, thus producing NO2 – abiotically. Because N2O was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO2 – is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO2 – enzymatically, but unlike cyt P460, its final product is NO, establishing it as an intermediate of nitrification. More broadly, NO can be recognized as a molecule common to the primary metabolisms of all organisms involved in nitrogen “defixation”. Delving deeper into cyt P460 yielded insights broadly applicable to controlled biochemical redox processes. Studies of an inactive cyt P460 from Nitrosomonas sp. AL212 showed that this enzyme was unable to oxidize NH2OH because it lacked a glutamate residue in its secondary coordination sphere that was present in the active N. europaea cyt P460 variant. Restoring the Glu residue imbued activity, revealing that a second-sphere base is Nature’s key to controlled oxidation of NH2OH. A key lesson of bioinorganic chemistry is reinforced: the polypeptide matrix is an essential part of dictating function. Our work also exposed some key functional contributions of noncanonical heme-protein cross-link...

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…Curiously, the Lancaster group observed similar, NO-dependent kinetics for the conversion of a 6C Fe­(II)–NO complex to the corresponding 5C species in the enzyme Cyt. P460 . Cyt.…”

Development of Nitrogen Recycling Strategies for Bioregenerative Life Support Systems in Space

“…Recently, however, nitric oxide (NO) has been demonstrated to be the enzymatic product of HAO instead of NO 2 − . Meanwhile, the production of NO 2 -from NO is probably catalyzed by a third, unidentified enzyme in the nitritation ( Coleman and Lancaster, 2020 ). The presence of such an enzyme is necessary to outcompete the side reactions that can produce by-products, such as NO3-and/or N 2 O, but also to prevent the spontaneous formation of NO 2 − .…”

“…The latter is required since non-enzymatic oxidation implies a loss of electrons, which are captured during the enzymatic reaction ( Caranto and Lancaster, 2018 ). Finally, NO 2 -is oxidized to NO3-by the membrane-bound nitrite oxidoreductase (NXR) of N. winogradskyi during the so-called nitratation (2), also using O 2 as an electron acceptor ( Starkenburg et al, 2006 ; Cruvellier et al, 2016 ; Caranto and Lancaster, 2018 ; Coleman and Lancaster, 2020 ).…”

Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen

“…In aerobic and anaerobic microbes, especially those of the biological nitrogen and sulfur cycles, there are many c -type cytochromes with multiple heme centers per polypeptide chain [ 2 , 3 , 5 , 23 , 25 – 32 , 35 , 55 , 98 , 103 , 114 – 122 ]. Early examples include the octaheme hydroxylamine oxidoreductase (HAO) and related proteins [ 123 – 129 ], the tetraheme NapC/NirT/TorC family [ 130 ], the 16-heme-containing protein Hmc (high molecular mass cytochrome c ) from sulfate-reducing bacteria [ 131 , 132 ], the octaheme tetrathionate reductase from Shewanella oneidensis [ 133 , 134 ], the tetraheme cytochrome c 3 described in the previous section, and the pentaheme cytochrome c nitrite reductase, the central enzyme of this review. These multiheme proteins form structurally related families, in which the positions of the heme can often be overlaid, even when there is little sequence conservation between members of the family, e.g., pentaheme nitrite reductase NrfA, octaheme HAO, and flavocytochrome fumarate reductase [ 22 , 133 – 135 ].…”