Nontypeable Haemophilus influenzae (NTHi) is a human pathogen implicated in infections and exacerbations of chronic obstructive pulmonary disease (COPD). Recent genomic mining efforts revealed a novel virulence factor, designated HvfA. Our laboratory established that HvfA is a ribosomally synthesized, posttranslationally modified peptide natural product (RiPP). The HvfA precursor peptide is expressed from the hvf biosynthetic gene cluster, which also encodes HvfB, a multinuclear nonheme iron-dependent oxidase (MNIO), and HvfC, a RiPP recognition element (RRE)-containing partner protein. The MNIO and its partner protein form a heterodimer that binds the precursor peptide and performs six posttranslational modifications of cysteine residues within HvfA to oxazolone and thioamide groups, yielding the mature virulence factor. Spectroscopic characterization has shown that HvfB contains a trinuclear iron cofactor that is likely responsible for cysteine oxidation, but the structure of the MNIO and its unusual metal cofactor remains unknown. X-ray crystallographic studies have proven intractable due to persistent issues with protein crystallization, so alternative measures have been taken to optimize the MNIO for structural determination by cryogenic electron microscopy (cryoEM). We engineered a terminus-to-terminus genetic fusion (HvfBCfusion) of the MNIO and the partner protein that replicates the native HvfBC heterodimer, enhances catalytic activity, and, importantly, increases the molecular weight to within practical range for cryoEM. Current work focuses on further optimization of HvfBCfusion for cryoEM analysis by integrating a rigid, nanobody-based LegoBody protein scaffold. Employing this macromolecule “carrier” scaffolding system overcomes the molecular weight barrier that excludes small proteins from cryoEM while also circumventing the prototypical structural biology bottleneck of protein crystallization. Determining the structure of HvfBCfusion will offer new insights into the emerging MNIO family of enzymes involved in the production of diverse RiPP natural products.