Metal Ion Selectivity and Substrate Inhibition in the Metal Ion Chelation Catalyzed by Human Ferrochelatase*

Protoporphyrin IX ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Ferrochelatase shows specificity, in vitro, for multiple metal ion substrates and exhibits substrate inhibition in the case of zinc, copper, cobalt, and nickel. Zinc is the most biologically significant of these; when iron is depleted, zinc porphyrins are formed physiologically. Examining the kcat/Kmapp ratios for zinc and iron reveals that, in vitro, zinc is the preferred substrate at all concentrations of porphyrin. This is not the observed biological specificity, where zinc porphyrins are abnormal; these data argue for the existence of a specific iron delivery mechanism in vivo. We demonstrate that zinc acts as an uncompetitive substrate inhibitor, suggesting that ferrochelatase acts via an ordered pathway. Steady-state characterization demonstrates that the apparent kcat depends on zinc and shows substrate inhibition. Although porphyrin substrate is not inhibitory, zinc inhibition is enhanced by increasing porphyrin concentration. This indicates that zinc inhibits by binding to an enzyme-product complex (EZnDIX) and is likely to be the second substrate in an ordered mechanism. Our analysis shows that substrate inhibition by zinc is not a mechanism that can promote specificity for iron over zinc, but is instead one that will reduce the production of all metalloporphyrins in the presence of high concentrations of zinc

Multi-locus sequence typing of Ixodes ricinus and its symbiont Candidatus Midichloria mitochondrii across Europe reveals evidence of local co-cladogenesis in Scotland.

Ticks have relatively complex microbiomes, but only a small proportion of the bacterial symbionts recorded from ticks are vertically transmitted. Moreover, co-cladogenesis between ticks and their symbionts, indicating an intimate relationship over evolutionary history driven by a mutualistic association, is the exception rather than the rule. One of the most widespread tick symbionts is Candidatus Midichloria, which has been detected in all of the major tick genera of medical and veterinary importance. In some species of Ixodes, such as the sheep tick Ixodes ricinus (infected with Candidatus Midichloria mitochondrii), the symbiont is fixed in wild adult female ticks, suggesting an obligate mutualism. However, almost no information is available on genetic variation in Candidatus M. mitochondrii or possible co-cladogenesis with its host across its geographic range. Here, we report the first survey of Candidatus M. mitochondrii in I. ricinus in Great Britain and a multi-locus sequence typing (MLST) analysis of tick and symbiont between British ticks and those collected in continental Europe. We show that while the prevalence of the symbiont in nymphs collected in England is similar to that reported from the continent, a higher prevalence in nymphs and adult males is apparent in Wales. In general, Candidatus M. mitochondrii exhibits very low levels of sequence diversity, although a consistent signal of host-symbiont coevolution was apparent in Scotland. Moreover, the tick MLST scheme revealed that Scottish specimens form a clade that is partially separated from other British ticks, with almost no contribution of continental sequence types in this north-westerly border of the tick's natural range. The low diversity of Candidatus M. mitochondrii, in contrast with previously reported high rates of polymorphism in I. ricinus mitogenomes, suggests that the symbiont may have swept across Europe recently via a horizontal, rather than vertical, transmission route

Feline acute intermittent porphyria: a phenocopy masquerading as an erythropoietic porphyria due to dominant and recessive hydroxymethylbilane synthase mutations

Human acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is an autosomal dominant inborn error of heme biosynthesis due to the half-normal activity of hydroxymethylbilane synthase (HMB-synthase). Here, we describe the first naturally occurring animal model of AIP in four unrelated cat lines who presented phenotypically as congenital erythropoietic porphyria (CEP). Affected cats had erythrodontia, brownish urine, fluorescent bones, and markedly elevated urinary uroporphyrin (URO) and coproporphyrin (COPRO) consistent with CEP. However, their uroporphyrinogen-III-synthase (URO-synthase) activities (deficient in CEP) were normal. Notably, affected cats had half-normal HMB-synthase activities and elevated urinary 5-aminolevulinic acid (ALA) and porphobilinogen (PBG), the deficient enzyme and accumulated metabolites in human AIP. Sequencing the feline HMB-synthase gene revealed different mutations in each line: a duplication (c.189dupT), an in-frame 3 bp deletion (c.842_844delGAG) identical to that causing human AIP and two missense mutations, c.250G>A (p.A84T) and c.445C>T (p.R149W). Prokaryotic expression of mutations c.842_844delGAG and c.445C>T resulted in mutant enzymes with <1% wild-type activity, whereas c.250G>A expressed a stable enzyme with ∼35% of wild-type activity. The discolored teeth from the affected cats contained markedly elevated URO I and III, accounting for the CEP-like phenocopy. In three lines, the phenotype was an autosomal dominant trait, while affected cats with the c.250G>A (p.A84T) mutation were homozygous, a unique recessive form of AIP. These animal models may permit further investigation of the pathogenesis of the acute, life-threatening neurological attacks in human AIP and the evaluation of therapeutic strategies. GenBank Accession Numbers: GQ850461–GQ850464

Cerebral venous thrombosis after vaccination against COVID-19 in the UK: a multicentre cohort study

BackgroundA new syndrome of vaccine-induced immune thrombotic thrombocytopenia (VITT) has emerged as a rare side-effect of vaccination against COVID-19. Cerebral venous thrombosis is the most common manifestation of this syndrome but, to our knowledge, has not previously been described in detail. We aimed to document the features of post-vaccination cerebral venous thrombosis with and without VITT and to assess whether VITT is associated with poorer outcomes.MethodsFor this multicentre cohort study, clinicians were asked to submit all cases in which COVID-19 vaccination preceded the onset of cerebral venous thrombosis, regardless of the type of vaccine, interval between vaccine and onset of cerebral venous thrombosis symptoms, or blood test results. We collected clinical characteristics, laboratory results (including the results of tests for anti-platelet factor 4 antibodies where available), and radiological features at hospital admission of patients with cerebral venous thrombosis after vaccination against COVID-19, with no exclusion criteria. We defined cerebral venous thrombosis cases as VITT-associated if the lowest platelet count recorded during admission was below 150 × 109 per L and, if the D-dimer was measured, the highest value recorded was greater than 2000 μg/L. We compared the VITT and non-VITT groups for the proportion of patients who had died or were dependent on others to help them with their activities of daily living (modified Rankin score 3–6) at the end of hospital admission (the primary outcome of the study). The VITT group were also compared with a large cohort of patients with cerebral venous thrombosis described in the International Study on Cerebral Vein and Dural Sinus Thrombosis.FindingsBetween April 1 and May 20, 2021, we received data on 99 patients from collaborators in 43 hospitals across the UK. Four patients were excluded because they did not have definitive evidence of cerebral venous thrombosis on imaging. Of the remaining 95 patients, 70 had VITT and 25 did not. The median age of the VITT group (47 years, IQR 32–55) was lower than in the non-VITT group (57 years; 41–62; p=0·0045). Patients with VITT-associated cerebral venous thrombosis had more intracranial veins thrombosed (median three, IQR 2–4) than non-VITT patients (two, 2–3; p=0·041) and more frequently had extracranial thrombosis (31 [44%] of 70 patients) compared with non-VITT patients (one [4%] of 25 patients; p=0·0003). The primary outcome of death or dependency occurred more frequently in patients with VITT-associated cerebral venous thrombosis (33 [47%] of 70 patients) compared with the non-VITT control group (four [16%] of 25 patients; p=0·0061). This adverse outcome was less frequent in patients with VITT who received non-heparin anticoagulants (18 [36%] of 50 patients) compared with those who did not (15 [75%] of 20 patients; p=0·0031), and in those who received intravenous immunoglobulin (22 [40%] of 55 patients) compared with those who did not (11 [73%] of 15 patients; p=0·022).InterpretationCerebral venous thrombosis is more severe in the context of VITT. Non-heparin anticoagulants and immunoglobulin treatment might improve outcomes of VITT-associated cerebral venous thrombosis. Since existing criteria excluded some patients with otherwise typical VITT-associated cerebral venous thrombosis, we propose new diagnostic criteria that are more appropriate