Ancient hemes for ancient catalysts

Tetrapyrroles are essential molecules in living organisms and perform a multitude of functions in all kingdoms. Their synthesis is achieved in cells via a complex biosynthetic machinery which is unlikely to be maintained, if unnecessary. Here we propose that ancient hemes, such as the d₁-heme of cd₁ nitrite reductase or the siroheme of bacterial and plant nitrite and sulphite reductases, are molecular fossils which have survived the evolutionary pressure because their role is strategic for the organism where they are found today. The peculiar NO-releasing propensity of the d₁-heme of P. aeruginosa NIR, recently shown by our group is, in our opinion, an example of this strategy. The hypothesis is that the d₁-heme structure might be a pre-requisite for the fast rate of NO dissociation from the ferrous form, a property which is crucial to enzymatic activity and cannot be achieved with a more common b-type heme.Key words: d1-heme, porphyrin, siroheme, nitrite reductase, sulphite reductase, nitric oxide, evolutionPseudomonas aeruginosa is a Gram-negative bacterium commonly found in soil and water, well known for its metabolic versatility; under anaerobic conditions it can use nitrate and nitrite to produce energy via the denitrification pathway. In natural environments, denitrification is the part of the biological nitrogen cycle in which nitrate is transformed into nitrogen gas; reduction of nitrate occurs in four stages each catalyzed by a specific metalloenzyme.^1,2 P. aeruginosa is also an opportunistic pathogen, capable of causing serious infections in several hosts, such as humans and plants^3,4; pathogenesis, NO metabolism and denitrification are strictly related.^5,6The conversion of nitrite (NO₂^-) to nitric oxide (NO) is catalyzed in denitrifying bacteria by the periplasmic nitrite reductases (NIR).⁷ In P. aeruginosa NIR is a heme-containing enzyme (cd₁NIR) which produces NO in the active site where the unique d₁-heme cofactor (Fig. 1) is bound. This peculiar heme is synthesized from iron-protoporphyrin IX and belongs to the isobacteriochlorines subgroup;¹ it is exclusively found in this type of bacterial NIR.Open in a separate windowFigure 1Chemical structure of the d₁-heme.Reduction of nitrite involves binding of this molecule to the reduced d₁-heme, followed by dehydration to yield NO; release of NO and re-reduction of the enzyme close the cycle. An high affinity for nitrite (and anions) of the ferrous d₁-heme is a peculiar feature of cd₁NIR.⁷ However since the product NO is a powerful inhibitor of ferrous hemeproteins, enzymatic turnover demands the quick release of NO. In our recent paper⁸ we have shown that NO dissociates rapidly from the reduced form of the specialized d₁-heme of P. aeruginosa cd₁NIR. This unexpected result indicates that cd₁NIR behaves differently from other hemeproteins, since the rate of NO dissociation is by far faster (more than 100-fold) than that measured for any other heme in the ferrous state.^8–11Our hypothesis is that the d₁-heme structure might be a prerequisite for the fast rate of NO dissociation from the ferrous form, a property which cannot be achieved with a standard b-type heme.A major consequence of our finding is that this property of the d₁-heme is essential to avoid quasi-irreversible binding of NO to the reduced heme, which would jeopardize the physiological function of the enzyme evolved to scavenge nitrite, the toxic product of nitrate reduction. From the bioenergetic view-point, the main energy-generating step in denitrification is nitrate reduction (with a net H⁺ traslocation of 2H⁺/2e^-); thus, although a complex electron transfer chain is often present, the major biological role of the reductive steps downstream of nitrate reduction is likely to be nitrite scavenging.² If the complex of NO with reduced cd₁NIR was very long lived it would hamper further reaction cycles thus resulting in the accumulation of nitrite which is toxic for the bacterium. In line with this interpretation, we have also shown very recently¹² that nitrite is able to displace NO from the ferrous enzyme; thus substrate availability is the key factor that controls the enzyme turnover.From the standpoint of molecular evolution it is accepted that bacterial denitrification is an ancient metabolic pathway which existed even before oxygen became abundant in the athmosphere. Several reports pointed out that the enzymes involved in aerobic respiration derive from those involved in the denitrification pathway. Primitive denitrifying bacteria (similar to the extant Paracoccus denitrificans) can be considered as a common ancestral symbiotic prototype of the eukaryotic mitochondrion. Indeed there is compelling evidence that modern eukaryotic oxidases evolved from bacterial NO-reductase once oxygen became available as a major oxidant.^13,14In microrganisms, other “ancient” metabolisms are represented by sulphite and nitrite reduction pathways, which were well suited for a prebiotic photoreducing environment.¹⁵ Also in these pathways several enzymes are heme-containing proteins in which modified hemes, such as siroheme, are used as cofactors.¹⁶ Interestingly also in plants siroheme is a relevant porphyrin group,¹⁷ being the cofactor of plant nitrite and sulphite reductases, required for the assimilation of inorganic nitrogen and sulphur from the environment.Tetrapyrroles are essential molecules in living organisms and perform a multitude of functions in all kingdoms. Their biosynthesis is achieved in cells via branched pathways which are expensive in terms of energy consumption.^16–18 The single pathways are tightly regulated and often activated only “on demand” when the specific heme group is required. Therefore, parsimony suggests that a complex biosynthetic machinery is unlikely to be maintained, if unnecessary.We thus propose that these ancient hemes (such as the d₁-heme or the siroheme) are molecular fossils which have survived the evolutionary pressure because their role is strategic only for the organism where they are found today. The peculiar NO-releasing propensity of the d₁-heme of P. aeruginosa NIR shown by our group could be, in our opinion, an example of this strategy. A major challenge for the future is to unveil other uncommon features of these hemes.