Heme Research Articles

Extremophilic hemoglobins: The structure of Shewanella benthica truncated hemoglobin N.

Truncated hemoglobins (TrHbs) have an ancient origin and are widely distributed in microorganisms where they often serve roles other than dioxygen transport and storage. In extremophiles, these small heme proteins must have features that secure function under challenging conditions: at minimum, they must be folded, retain the heme group, allow substrates to access the heme cavity, and maintain their quaternary structure if present and essential. The genome of the obligate psychropiezophile Shewanella benthica strain KT99 harbors a gene for a TrHb belonging to a little-studied clade of globins (subgroup 2 of group N). In the present work, we characterized the structure of this protein (SbHbN) with electronic absorption spectroscopy and X-ray crystallography, and inspected its structural integrity under hydrostatic pressure with NMR spectroscopy and small-angle X-ray scattering. We found that SbHbN self-associates weakly in solution and contains an extensive network of hydrophobic tunnels connecting the active site to the surface. Amino acid replacements at the dimeric interface formed by helices G and H in the crystal confirmed this region to be the site of intermolecular interactions. High hydrostatic pressure dissociated the assemblies while the porous subunits resisted unfolding and heme loss. Preservation of structural integrity under pressure is also observed in non-piezophilic TrHbs, which suggests that this ancient property is derived from functional requirements. Added to the inability of SbHbN to combine reversibly with dioxygen and a propensity to form heme d, the study broadens our perception of the TrHb lineage and the resistance of globins to extreme environmental conditions.

Coordination of inorganic disulfide species to ferric N-acetyl microperoxidase 11.

The interest in chemical interactions between inorganic sulfur species and heme compounds has grown significantly in recent years due to their physiological relevance. The model system ferric N-acetyl microperoxidase 11 (NAcMP11FeIII) enables the exploration of the mechanistic aspects of the interaction between the ferric heme group and binding sulfur ligands, without the constraints imposed by a protein matrix and the stabilizing effects of distal amino acids. In this study, we investigated the coordination of disulfane (HSSH) and its conjugate base hydrodisulfide (HSS-) to NAcMP11FeIII. Kinetic estimations of the binding constant retrieved a pH-independent kon= (1.5±0.7) x105M-1s-1, for 6.4≤pH≤7.2, and a similar value for the intrinsic constant for HSS-, the predominant species. To obtain a molecular description of the binding process, we resorted to two complementary theoretical approaches. Firstly, using multiple steered molecular dynamics, we calculated the free energy profiles for the migration of the neutral species HSSH and the monoanionic HSS-, and also for the siblings hydrogen sulfide, H2S, and hydrosulfide, HS-. Our results reveal that both neutral and anionic species can achieve the distal cavity, as expected considering the highly solvent exposed heme group in NAcMP11FeIII. Secondly, we explored the ligand-exchange reaction using a combination of nudged elastic band (NEB) and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations, which suggest that the monoanionic species can displace the water molecule coordinated to the heme iron more efficiently than the neutral ones. Altogether, our results provide a molecular description of the ligand binding process of these sulfur species to ferric heme proteins.

A Closer Look at the FeS Heme Bonds in Azotobacter vinelandii Bacterioferritin: QM/MM and Local Mode Analysis

ABSTRACTUsing the QM/MM methodology and a local mode analysis, we investigated a character and a strength of FeS bonds of heme groups in oxidized and reduced forms of Bacterioferritin from Azotobacter vinelandii. The strength of the FeS bonds was correlated with a bond length, an energy density at a bond critical point, and a charge difference of the F and S atoms. Changing the oxidation state from ferrous to ferric generally makes the FeS bonds weaker, longer, more covalent, and more polar. We also investigated the SFeS bond bending and found that the stronger FeS bond, generally makes the SFeS bond bending stiffer, which could play a key role in the balance between ferric and ferrous oxidation states and related biological activities.

In vitro studies on the metal sensing capability of annelide hemoglobin

Many studies in the literature suggest that earthworms play an important role in the use and management of soils and are considered good bioindicators of environmental contamination by presenting changes in their behavior and even death when inserted in contaminated environments. In the current study we investigated the in vitro behavior of Amynthas gracilis hemoglobin (HbAg) against copper (Cu) and cadmium (Cd) in concentrations ranging from 0 to 100 µmol L-1, at pH values 5.0 and 7.0 by optical absorption, fluorescence emission, light scattering and circular dichroism techniques. Our results sugest that the HbAg undergoes oxidation in its heme group at both pH values. As from 30 µmol L-1 of copper in pH 7.0 HbAg is oxidized, dissociates and loses part of its secondary structure. In pH 5.0, it undergoes greater oxidation, also near 30 µmol L-1 but without secondary structures loss or dissociation. Regarding the metal cadmium, no significant changes were observed. The results indicate that HbAg has a potential for studies on copper biosensing.

New insights in uranium bioremediation by cytochromes of the bacterium Geotalea uraniireducens.

The bacterium Geotalea uraniireducens, commonly found in uranium-contaminated environments, plays a key role in bioremediation strategies by converting the soluble hexavalent form of uranium (U(VI)) into less soluble forms (e.g., U(IV)). While most of the reduction and concomitant precipitation of uranium occur outside the cells, there have been reports of important reduction processes taking place in the periplasm. In any case, the triheme periplasmic cytochromes are key players, either by ensuring an effective interface between the cell's interior and exterior or by directly participating in the reduction of the metal. Therefore, understanding the functional mechanism of the highly abundant triheme cytochromes in G.uraniireducens' is crucial for elucidating the respiratory pathways in this bacterium. In this work, a detailed functional characterization of the triheme cytochromes PpcA and PpcB from G.uraniireducens was conducted using NMR and visible spectroscopy techniques. Despite sharing high amino acid sequence identity and structural homology with their counterparts from Geobacter sulfurreducens, the results showed that the heme reduction potential values are less negative, the order of oxidation of the hemes is distinct, and the redox and redox-Bohr network of interactions revealed unprecedented functional mechanisms in the cytochromes of G.uraniireducens. In these cytochromes, the reduction potential values of the three heme groups are much more similar, resulting in a narrower range of values, that facilitates directional electron flow from the inner membrane, thereby optimizing the uranium reduction.

Computational journey to unveil organophosphorothioate pesticides’ metabolism: A focus on chlorpyrifos and CYP2C19 mutational landscape

Organophosphorothioates (OPT) are pesticides impacting human, animal and environmental health. They enter the environment worldwide, primarily due to their application as insecticides. OPTs are mainly neurotoxic upon bioactivation and inhibition of brain and serum acetylcholinesterase (AChE). Although OPTs are meant to target insects, they are potentially toxic to many other species (including humans), posing risks to non-target organisms and ecosystems. Certain cytochromes P450 (CYP) promote OPTs bioactivation, forming the corresponding oxon metabolites, while others catalyse their detoxification. Understanding the molecular basis of such a bivalent fate may help to clarify the toxicity of OPTs in living organisms, with far-reaching consequences to understand their impact on living organisms and improve risk assessment, to cite but a few. However, although crucial, the underpinning mechanisms still lay unclear. Here, a validated computational pipeline revealed the molecular reasons underlying the differential metabolism of chlorpyrifos in humans by CYP2C19, a primal route of detoxification, and its bioactivation by CYP2B6. The analysis drew the diverse occupancy of the CYP pocket and orientation to the heme group as a convincing evidence-based explanation for the opposite transformation. Moreover, this study explored the impact of CYP2C19 mutational landscape giving a blueprint to unveil the molecular basis of OPTs metabolism and toxicological implications from an inter-individual perspective. Taken together, the outcome described for the first time to the best of our knowledge a structural rationale for the bioactivation/detoxification of OPTs improving the current understanding of their toxicity from a molecular standpoint.

Identification and quantification of the molecular species of bilirubin BDG, BMG and UCB by LC‒MS/MS in hyperbilirubinemic human serum.

Unconjugated bilirubin (UCB) is a byproduct of the heme group that indicates irregularities in the metabolism of several important biological molecules, such as hemoglobin. UCB is processed by hepatic UGT1A1, which catalyzes its conjugation to the metabolites bilirubin diglucuronide (BDG) and bilirubin monoglucuronide (BMG). The serum concentrations of BDG and BMG may indicate liver injury or dysfunction. The aim of this study was to standardize and validate a method for the identification and simultaneous quantification of BMG, BDG and UCB by LC‒MS/MS. Liquid‒liquid extraction allows the separation of UCB, BMG and BDG from the serum of healthy subjects or patients with liver injury. Detection and quantification were performed using an LC‒MS/MS method. Compound separation was achieved with a BEH-C18 column at 40°C. The mobile phase was prepared with 5 mM ammonium acetate (pH 6) and acetonitrile, and a flow gradient was applied. This is the first study to directly quantify BMG and UCB levels in human serum; no postcalculations or correction factors are needed. However, BDG quantification requires calculations and a correction factor. We identified the molecular species with ionic transitions m/z1+ 585.4 > 299.2 for UCB, 761.3 > 475.3 for BMG, 937.3 > 299.5 for BDG and mesobilirubin 589.4 > 301.3 (IS). The procedures used in this study allowed the simultaneous identification and quantification of the molecular species of bilirubin, BDG, BMG and UCB. Analysis of the serum levels in patients with hyperbilirubinemia revealed that patients with acute-on-chronic liver failure had elevated levels of these species.

Catalase‐Based Air‐Cathode for Biocompatible Zinc–Air Battery

AbstractThe use of catalase (CAT) from bovine liver as an electrocatalyst in the air electrode of a primary zinc–air battery (ZAB) with neutral electrolyte (pH 7.4) is reported. First, the use of Nafion for immobilizing CAT onto electrode surface allowed to obtain long‐term stability of the catalytic activity of CAT. Besides, analysis of the CAT and CAT‐Nafion as catalysts for oxygen reduction reaction (ORR) was carried out by linear sweep voltammetry (LSV) using a rotating ring‐disk electrode (RRDE), demonstrating a four‐electron ORR mechanism. Finally, CAT and CAT‐Nafion were tested as air electrodes in flooded ZAB and compared against human hemoglobin‐based ZAB. Our results demonstrated the significant influence of the protein coating of the heme groups on the ZAB performance, affecting greatly the discharge capacity and the power density.

Heme catabolism and heme oxygenase-1-expressing myeloid cells in pathophysiology.

Although the pathological significance of myeloid cell heterogeneity is still poorly understood, new evidence indicates that distinct macrophage subsets are characterized by specific metabolic programs that influence disease onset and progression. Within this scenario, distinct subsets of macrophages, endowed with high rates of heme catabolism by the stress-responsive enzyme heme oxygenase-1 (HO-1), play critical roles in physiologic and pathological conditions. Of relevance, the substrates of HO-1 activity are the heme groups that derive from cellular catabolism and are converted into carbon monoxide (CO), biliverdin and Fe2+, which together elicit anti-apoptotic, anti-inflammatory activities and control oxidative damage. While high levels of expression of HO-1 enzyme by specialized macrophage populations (erythrophagocytes) guarantee the physiological disposal of senescent red blood cells (i.e. erythrocateresis), the action of HO-1 takes on pathological significance in various diseases, and abnormal CO metabolism has been observed in cancer, hematological diseases, hypertension, heart failure, inflammation, sepsis, neurodegeneration. Modulation of heme catabolism and CO production is therefore a feasible therapeutic opportunity in various diseases. In this review we discuss the role of HO-1 in different pathological contexts (i.e. cancer, infections, cardiovascular, immune-mediated and neurodegenerative diseases) and highlight new therapeutic perspectives on the modulation of the enzymatic activity of HO-1.

Mechanisms of the Formation and Function of Dinitrosyl Iron Complexes as a “Working Form” of Nitric Oxide in Living Organisms

It has been proposed that only the introduction of endogenous nitric oxide (NO) into dinitrosyl iron complexes (DNIC) or S-nitrosothiols (RS-NO) can ensure stabilization of NO that is critical for operating in an auto/paracrine manner as a regulator of biological processes in living organisms. Without this introduction, the majority of endogenous NO disappears due to aggressive intra/intercellular environment thereby eliminating it from metabolic processes. Administration of exogenous NO in human and animal organisms (the only possible route of administration is via inhalation when it is in the gaseous state) does not lead to formation of DNIC or RS-NO in blood or other tissues. Hence, the majority of exogenous NO during its inhalation is converted into nitrosonium cations (NO+), the emergence of which is evidenced by their conversion into RS-NO when various thiol-containing compounds are administered to animal blood concomitantly. In turn, RS-NO formation in those animals was manifested by hypotensive effect on them. NO molecule transformation into nitrosonium cations may also occur during DNIC formation induced by a disproportionation reaction of endogenous NO molecules binding with Fe2+ ions in pairs. Subsequent binding of Fe(NO)2 groups to thiol-containing ligands that occur during this reaction promotes formation of rather stable DNICs that act as donors of both neutral molecules of NO and nitrosonium cations (NO+) in living organisms. The transfer of NO and NO+ to targets for nitrosation reactions occurs by the interaction of low molecular DNICs with heme groups of heme-containing proteins (for example, guanylate cyclase) or thiol groups in low molecular or protein thiol-containing compounds. This paper presents different results of NO and NO+ transfer in living organisms disussing both positive, regulatory and negative, toxic effects of it.

Simultaneous Reduction and Methylation of Nanoparticulate Mercury: The Critical Role of Extracellular Electron Transfer.

Mercury nanoparticles are abundant in natural environments. Yet, understanding their contribution to global biogeochemical cycling of mercury remains elusive. Here, we show that microbial transformation of nanoparticulate divalent mercury can be an important source of elemental and methylmercury.Geobacter sulfurreducensPCA, a model bacterium predominant in anoxic environments (e.g., paddy soils), simultaneously reduces and methylates nanoparticulate Hg(II). Moreover, the relative prevalence of these two competing processes and the dominant transformation pathways differ markedly between nanoparticulate Hg(II) and its dissolved and bulk-sized counterparts. Notably, even when intracellular reduction of Hg(II) nanoparticles is constrained by cross-membrane transport (a rate-limiting step that also regulates methylation), the overall Hg(0) formation remains substantial due to extracellular electron transfer. With multiple lines of evidence based on microscopic and electrochemical analyses, gene knockout experiments, and theoretical calculations, we show that nanoparticulate Hg(II) is preferentially associated with c-type cytochromes on cell membranes and has a higher propensity for accepting electrons from the heme groups than adsorbed ionic Hg(II), which explains the surprisingly larger extent of reduction of nanoparticles than dissolved Hg(II) at relatively high mercury loadings. These findings have important implications for the assessment of global mercury budgets as well as the bioavailability of nanominerals and mineral nanoparticles.

Hooked on fish blood: the reliance of a gill parasite on haematophagy.

Parasitism involves diverse evolutionary strategies, including adaptations for blood feeding, which provides essential nutrients for growth and reproduction. Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), an ectoparasitic flatworm, infects the gills of gilthead seabream (Sparus aurata), significantly affecting fish health, welfare and Mediterranean cage farm profitability. Despite its impact, limited information exists on its feeding behaviour. This study demonstrates the presence of blood and exogenous haem groups in S. chrysophrii and explores its digestive tract using light and electron microscopy, elucidating its internal morphology and spatial arrangement. Elemental analysis of the digestive haematin cells shows residual oxidized haem depots as haematin crystals. Additionally, we studied the impact of the blood feeding on the host by estimating the average volume of blood intake for an adult parasite (2.84 ± 2.12µl·24h-1) and we described the significant drop of the plasmatic free iron levels in infected hosts. Overall, we demonstrate the parasite's reliance on its host blood, the parasite's buccal and digestive morphological adaptations for blood feeding and the provoked effect on the fish host's health.

Food Supplement Based on L-Arginine and Grape Extract for the Control of Blood Pressure

The treatment of most patients with elevated blood pressure requires drug therapy, as well as lifestyle measures to achieve optimal blood pressure control. There are numerous side effects resulting from the use of these drugs: from the persistent dry cough of ACE inhibitors, to the headache, dizziness and tiredness of CCBs, to the slow heartbeat and sleep disturbances of betablockers. Scientific research in recent years is focusing attention on safer alternative therapeutic remedies, possibly of natural origin, also in support of classic pharmacological therapies, in order to reduce the doses of active ingredients and, consequently, their side effects. The present project aims to clinically test a nutraceutical product consisting of a formulation based on L-arginine and grape pomace extract of the Aglianico cultivar microencapsulated into maltodextrin. The formulation acts by: favoring the NO-soluble GC (sGC)-cGMP pathway; with an antioxidant effect on the heme group Fe+2, with a greater production of cGMP. The study involves the enrollment of 80 patients in 3 internal medicine units adhering to FADOI Campania. The treatment involves 1 tablet/ day. Project duration for each UOC: 80 days.

Comparative analysis of the interaction mechanism of γ-globulin and hemoglobin with spherical and rod-shaped gold nanoparticles

The interaction mechanism of spherical gold nanoparticles (AuNPs) and rod-shaped gold nanoparticles (AuNRs) with γ-globulin and hemoglobin was comprehensively and comparatively analyzed. γ-Globulin and hemoglobin have high affinity with AuNPs, and with two different types of binding sites on AuNRs surface. Except hemoglobin interaction with the first binding site of AuNRs, the interaction between γ-globulin/hemoglobin and AuNPs/AuNRs is the spontaneous, endothermic and entropy-driven process, and hydrophobic interaction plays a dominant role. The molecular adsorption mechanism of γ-globulin/hemoglobin on AuNPs and AuNRs surface conforms to Langmuir model and Freundlich model, respectively. The kinetic molecular mechanism between them conforms to the pseudo-second-order model, and chemisorption is the rate-limiting step. AuNPs result in the loosening and unfolding of γ-globulin backbone. AuNRs have no significant effect on γ-globulin backbone. AuNPs/AuNRs result in no significant changes in hemoglobin structure and heme group microenvironment. AuNPs/AuNRs decrease the hydrophobicity of Trp microenvironment of γ-globulin, but there is an intramolecular energy transfer from Trp residue to Tyr residue of hemoglobin. The β-sheet of γ-globulin and the α-helix of hemoglobin reduce by increasing concentrations of AuNPs/AuNRs. Molecular docking is suggesting that the specific amino acid residues of γ-globulin and hemoglobin interaction with AuNPs/AuNRs, and validates the experimental results.

Combined fluorometric analysis of biliverdin and bilirubin by the recombinant protein HUG

Biliverdin is a secondary metabolite of heme catabolism. It is formed by the reaction catalyzed by heme oxygenase, which converts the heme group contained in proteins such as hemoglobin, myoglobin, cytochromes, and catalase into biliverdin, iron (II) and CO in equimolar amounts, consuming NADPH. Biliverdin is then reduced to bilirubin by biliverdin reductase. Biliverdin and bilirubin form a redox couple and are important for the redox homeostasis of cells. Heme oxygenase-1 is an inducible enzyme that is induced by hypoxic conditions, increased availability of heme or proinflammatory mechanisms such as LPS, UV radiation, etc. In addition, both heme oxygenase-1 and biliverdin reductase play roles other than catalysis by modulating specific metabolic pathways at the transcriptional level. There is a need for affordable assays to analyze these bile pigments in biological and clinical samples. Here we present a method for the combined determination of biliverdin and bilirubin that utilizes the specific binding of bilirubin to the fluorescent recombinant fusion protein HUG and the enzymatic conversion of biliverdin to bilirubin.•This method enables the combined measurement of bilirubin and biliverdin in the nM range.•The method does not require solvent extraction or protein precipitation of the samples.

Full-length structure and heme binding in the transcriptional regulator HcpR.

HcpR is a CRP-family transcriptional regulator found in many Gram-negative anaerobic bacteria. In the perio-pathogen Porphyromonas gingivalis, HcpR is crucial for the response to reactive nitrogen species such as nitric oxide (NO). Binding of NO to the heme group of HcpR leads to transcription of the redox enzyme Hcp. However, the molecular mechanisms of heme binding to HcpR remain unknown. In this study we present the 2.3Å structure of the P. gingivalis HcpR. Interdomain interactions present in the structure help to form a hydrophobic pocket in the N-terminal sensing domain. A comparison analysis with other CRP-family members reveals that the molecular mechanisms of HcpR-mediated regulation may be distinct from other family members. Using docking studies, we identify a putative heme binding site in the sensing domain. In vitro complementation and mutagenesis studies verify Met68 as an important residue in activation of HcpR. Finally, heme binding studies with purified forms of recombinant HcpR support Met68 and His149 residues as important for proper heme coordination in HcpR.

Rational design of 2-benzylsulfinyl-benzoxazoles as potent and selective indoleamine 2,3-dioxygenase 1 inhibitors to combat inflammation

Mimicking the transition state of tryptophan (Trp) and O2 in the enzymatic reaction is an effective approach to design indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors. In this study, we firstly assembled a small library of 2-substituted benzo-fused five membered heterocycles and found 2-sulfinyl-benzoxazoles with interesting IDO1 inhibitory activities. Next the inhibitory activity toward IDO1 was gradually improved. Several benzoxazoles showed potent IDO1 inhibitory activity with IC50 of 82–91 nM, and exhibited selectivity between IDO1 and tryptophan 2,3-dioxygenase (TDO2). Enzyme binding studies showed that benzoxazoles are reversible type II IDO1 inhibitors, and modeling studies suggested that the oxygen atom of the sulfoxide in benzoxazoles interacts with the iron atom of the heme group, which mimics the transition state of Fe-O-O-Trp complex. Especially, 10b can effectively inhibit the NO production in lipopolysaccharides (LPS) stimulated RAW264.7 cells, and it also shows good anti-inflammation effect on mice acute inflammation model of croton oil induced ear edema.

In Vitro Effect of Epigallocatechin Gallate on Heme Synthesis Pathway and Protoporphyrin IX Production.

Photodynamic therapy (PDT) treats nonmelanoma skin cancer. PDT kills cells through reactive oxygen species (ROS), generated by interaction among cellular O2, photosensitizer and specific light. Protoporphyrin IX (PpIX) is a photosensitizer produced from methyl aminolevulinate (MAL) by heme group synthesis (HGS) pathway. In PDT-resistant cells, PDT efficacy has been improved by addition of epigallocatechin gallate (EGCG). Therefore, the aim of this work is to evaluate the effect of EGCG properties over MAL-TFD and PpIX production on A-431 cell line. EGCG's role over cell proliferation (flow cytometry and wound healing assay) and clonogenic capability (clonogenic assay) was evaluated in A-431 cell line, while the effect of EGCG over MAL-PDT was determined by cell viability assay (MTT), PpIX and ROS detection (flow cytometry), intracellular iron quantification and gene expression of HGS enzymes (RT-qPCR). Low concentrations of EGCG (<50 µM) did not have an antiproliferative effect over A-431 cells; however, EGCG inhibited clonogenic cell capability. Furthermore, EGCG (<50 µM) improved MAL-PDT cytotoxicity, increasing PpIX and ROS levels, exerting a positive influence on PpIX synthesis, decreasing intracellular iron concentration and modifying HGS enzyme gene expression such as PGB (upregulated) and FECH (downregulated). EGCG inhibits clonogenic capability and modulates PpIX synthesis, enhancing PDT efficacy in resistant cells.

Exploring structural and computational contrasts in Myoglobins: Implications for thermal treatment-induced Sulfmyoglobin formation

Greening of tuna metmyoglobin (MetMb) by thermal treatment (TT) and free cysteine is associated with sulfmyoglobin (SulfMb) production. This greening reaction (GR) was once thought to occur only in tuna species. However, recent research has revealed that not all tuna species exhibit this behavior, and it can also occur in horse MetMb. Thus, the present study aimed to compare the GR-reactive (Katsuwonus pelamis and Equus caballus) and GR-unreactive (Sarda chiliensis and Euthynnus lineatus) MetMb using UV–vis spectrometry during TT (60 °C/30 min and free cysteine) to monitor the GR. We used molecular dynamics (MD) simulation to assess the stability of the heme group during TT. We discovered that using GR-unreactive MetMb resulted in an incomplete GR without producing SulfMb. Additionally, our MD simulations indicated that Met85 presence in the heme cavity from GR-unreactive is responsible for the heme group instability and displacement of distal His during TT.

Proton transfer in cytochrome bd-I from E. coli involves Asp-105 in CydB

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b558, heme b595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer.Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b558 is pH dependent, with a maximum rate constant (~104 s−1) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pKa ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58B➔Asn and Asp105B➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105B➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pKa proton donor, and the conservation pattern of the Asp-105B in the cyt. bd superfamily.