Heme edge reactivity towards sulfur- and oxygen-based stress agents



PD 101



Contracting authority:         Executive Unit for Financing Higher Education, Research, Development and Innovation (UEFISCDI)

Implementation period: 01/04/2022 - 31/03/2024

Budget:                         250.000 RON

Project director:           Dr. Eng. Adrian M.V. Brβnzanic

Mentor:                        Prof. Dr. Radu-Silaghi Dumitrescu




The project aims to explore, through means of molecular modelling methods, the formation edge-derived heme species in the active site of sulfite reductase. The enzyme was previously shown to use a modified version a heme, namely siroheme, in order to avoid electron transfer through inefficient routes. It was hypothesised that, beside providing a faster route for the transferred electrons required by catalysis, siroheme would also hinder the formation of edge-derived species, such as sulfheme and hydroxyheme, that are known to drastically affect the Fe atoms substrate binding affinity and that can cause porhyrin cleavage. Here we intend to prove that the special conditions provided by the sulfite reductase active site environment could lead to the formation edge-derived hemes through previously unknown mechanisms.



Project Goals

Can anionic heme radicals cause the formation of heme derived compounds in the presence of S- and/or solvent molecules? Beside charge transfer optimisation, can the SiR blockade also inhibit undesired side-reactions, or are these reactions impossible even in the absence of this blockade?  This is the core problem to which we aim to provide an answer.



Phase I

• DFT calculations on the sulfite reductase reaction mechanism to better understand the nature of the possible intermediates present and that have the potential to react with the porphyrin edge of the active site.

• Calculation of the most favorable electronic transfer routes in the synthetic version (i.e. heme-cuban) of the active site.

• Processing and preparing these results for publication.

• Preparation of the QM/MM system of sulfite reductase enzyme.

• Parameterization of the active site of the sulfite reductase enzyme.

• Performing QM/MM calculations on the inactive version (i.e. with phosphate bound to iron heme).


Phase II

• QM/MM calculations on all possible broken-symmetry solutions of phosphate bound model of siroheme-containing sulfite reductase.

• QM/MM calculations on biological variant (i.e. siroheme containing) models containing substrates relevant for the sulfite reductase reaction mechanism such S/O bound SO3,­ SO2, SO and SH in the presence of solvent (i.e. one water molecule).

• QM calculations of small heme models interacting with the thiyl radical.

• Construction of heme containing models for the synthetic version of sulfite reductase.

• QM/MM calculations on heme containing models of the synthetic version of sulfite reductase.


Phase III

In this step, QM/MM MD calculations were performed to elucidate the role of siroheme in avoiding unwanted side reactions in the active site of the sulfite reductase enzyme. Both siroheme and its fully conjugated variant, heme S, do not show a reactivity of the periphery of the porphyrin ring towards SH and OH radicals. Thus, the specific blockade of siroheme on the transfer of electrons from the cubane to the catalytic Fe does not seem to play an important role in avoiding secondary reactions.

The calculations were performed at the same DFT level (i.e. QM= BS-TPSS/def2-SV(P)-D3 and MM= ff14SB) but in MD (molecular dynamics) mode. 100 steps were calculated at an interval of 0.5 fs, using the Berendsen thermostat. All these QM/MM MD calculations were done with Orca software after the MM component was obtained from Amber.

Relevant results can be seen here:

·       Siroheme – SH: https://youtu.be/E1aVImzvnPs

·       Siroheme – OH: https://youtu.be/jATydnlpOTE

·       Heme S – SH: https://youtu.be/OZy8FQIfwDA

·       Heme S – OH: https://youtu.be/WQihmuN0J84



Thus, based on the results obtained so far, it can be concluded that the objectives of the project have been met, and the impact is estimated to be a major one in the field of bioinorganic chemistry. This major impact has already been highlighted by the presentations given at the two bioinorganic chemistry conferences (cf. EuroBIC 2022, Grenoble FRA - poster presentation - and ICBIC 2023, Adelaide AUS - oral presentation) given by the director of this project.


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