Heme redox potentials hold the key to reactivity differences between nitric oxide reductase and heme-copper oxidase

Abstract

Despite high structural homology between NO reductases (NORs) and heme-copper oxidases (HCOs), factors governing their reaction specificity remain to be understood. Using a myoglobin-based model of NOR (FeMb) and tuning its heme redox potentials (E°') to cover the native NOR range, through manipulating hydrogen bonding to the proximal histidine ligand and replacing heme with monoformyl (MF-) or diformyl (DF-) hemes, we herein demonstrate that the E°' holds the key to reactivity differences between NOR and HCO. Detailed electrochemical, kinetic, and vibrational spectroscopic studies, in tandem with density functional theory calculations, demonstrate a strong influence of heme E°' on NO reduction. Decreasing E°' from +148 to -130 mV significantly impacts electronic properties of the NOR mimics, resulting in 180- and 633-fold enhancements in NO association and heme-nitrosyl decay rates, respectively. Our results indicate that NORs exhibit finely tuned E°' that maximizes their enzymatic efficiency and helps achieve a balance between opposite factors: fast NO binding and decay of dinitrosyl species facilitated by low E°' and fast electron transfer facilitated by high E°'. Only when E°' is optimally tuned in FeMb(MF-heme) for NO binding, heme-nitrosyl decay, and electron transfer does the protein achieve multiple (>35) turnovers, previously not achieved by synthetic or enzyme-based NOR models. This also explains a long-standing question in bioenergetics of selective cross-reactivity in HCOs. Only HCOs with heme E°' in a similar range as NORs (between -59 and 200 mV) exhibit NOR reactivity. Thus, our work demonstrates efficient tuning of E°' in various metalloproteins for their optimal functionality.

Synthetic Fe/Cu Complexes: Toward Understanding Heme-Copper Oxidase Structure and Function

“…In this study, the results of various vibrational, spectroscopic, electrochemical, and computational methods performed on the different heme-containing model systems showed that these rate determining factors for NO reduction can be controlled by tuning heme E°ˊ to achieve ~35 turnovers (based on quantification of N 2 O production using GC/MS), which is more than any previous synthetic (small molecule) or engineered protein system. 1115 This result was obtained with the Fe B Mb(MF-heme) protein which contains a monoformylated (Figure 143) heme with E°' = +53 ± 7 mV versus SHE. The significance of these results is further relevant for understanding the even wider variation in functions observed with other heme proteins as well (O 2 -transporting globins, vs O 2 -activating HCOs and P450s, vs NORs), which all have evolutionarily tuned heme environments and redox potentials.…”

“…In the modified Mb models, by changing the identity of the incorporated heme or the H-bonding to the proximal His ligand (which effectively lowers the heme redox potential), 309 they were able to tune the heme iron E°' and, in doing so, observed a significant enhancenment in NOR activity at lower heme potentials. 1115 The key to NO reduction in NORs is balancing the energetic favorability of (i) NO binding, (ii) Fe-NO decay, and (iii) electron transfer to the (heme)Fe-NO intermediate, analogous to the necessity for HCOs to control O 2 binding, ROS release, and PCET to the bound oxy ligand. All of these processes are affected by heme redox potentials.…”

Mimochrome, a metalloporphyrin‐based catalytic Swiss knife†

“…Rational design of the Mb distal site has been adopted to install HCOlike [123,124] and NOR-like [125] active sites, adding a new mononuclear cofactor. These putative Cu B and Fe B sites, together with heme, were not only structural and functional mimetics but were helpful in elucidating key aspects about the activity of their natural counterparts [126][127][128]. More recently, cytochrome c peroxidase (CcP) activity was steered toward an active SiR surrogate by installing a [4Fe-4S] cofactor in its proximal site [129].…”

Activation of O2and NO in heme-copper oxidases – mechanistic insights from computational modelling

“…2). 11,124 In Fig. 25 (left part) the energy profile for mechanism I in cbb 3 oxidases is compared to the corresponding profile for the a 3 oxidases, starting from the nitrosyl complex.…”